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研究论文
Structure and Biophysics for a Six Letter DNA Alphabet that Includes Imidazo[1,2-a]-1,3,5-triazine-2(8H)-4(3H)-dione (X) and 2,4-Diaminopyrimidine (K)
包括咪唑[1,2-a]-1,3,5-三嗪-2（8H）-4（3H）-二酮 （X） 和 2,4-二氨基嘧啶 （K） 的六字母 DNA 字母表的结构和生物物理学Click to copy article link
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- Isha Singh
伊莎·辛格 - Myong-Jung Kim
金明贞 - Robert W. Molt
罗伯特·莫尔特 （Robert W. Molt - Shuichi Hoshika
Shuichi Hoshika 星香修一 - Steven A. Benner
史蒂文·本纳 - Millie M. Georgiadis
米莉·乔治亚迪斯
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† Department of Biochemistry & Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202, United States
‡ Foundation for Applied Molecular Evolution, and the Westheimer Institute of Science & Technology, 13709 Progress Boulevard, Box 7, Alachua, Florida 32615, United States
§ Firebird Biomolecular Sciences LLC, 13709 Progress Boulevard, Box 17, Alachua, Florida 32615, United States
⊥ ENSCO, Inc., 4849 North Wickham Road, Melbourne, Florida 32940, United States
¶ Department of Chemistry and Chemical Biology, Indiana University, Purdue University Indianapolis, Indianapolis, Indiana 46202, United States
*Tel.: (317) 278-8486. Fax: (317) 274-4686. E-mail: mgeorgia@iu.edu
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A goal of synthetic biology is to develop new nucleobases that retain the desirable properties of natural nucleobases at the same time as expanding the genetic alphabet. The nonstandard Watson–Crick pair between imidazo[1,2-a]-1,3,5-triazine-2(8H)-4(3H)-dione (X) and 2,4-diaminopyrimidine (K) does exactly this, pairing via complementary arrangements of hydrogen bonding in these two nucleobases, which do not complement any natural nucleobase. Here, we report the crystal structure of a duplex DNA oligonucleotide in B-form including two consecutive X:K pairs in GATCXK DNA determined as a host–guest complex at 1.75 Å resolution. X:K pairs have significant propeller twist angles, similar to those observed for A:T pairs, and a calculated hydrogen bonding pairing energy that is weaker than that of A:T. Thus, although inclusion of X:K pairs results in a duplex DNA structure that is globally similar to that of an analogous G:C structure, the X:K pairs locally and energetically more closely resemble A:T pairs.
合成生物学的一个目标是开发新的核碱基，在扩大遗传字母表的同时保留天然核碱基的理想特性。咪唑[1,2-a]-1,3,5-三嗪-2（8H）-4（3H）-二酮 （X） 和 2,4-二氨基嘧啶 （K） 之间的非标准 Watson-Crick 对正是这样做的，通过这两个核碱基中的氢键互补排列配对，它们不补充任何天然核碱基。在这里，我们报道了 B 型双链 DNA 寡核苷酸的晶体结构，包括 GATCXK DNA 中的两个连续 X：K 对，以 1.75 Å 分辨率测定为主客体复合物。X：K 对具有显着的螺旋桨扭转角，类似于观察到的 A：T 对，并且计算出的氢键对能比 A：T 弱。因此，尽管包含 X：K 对会导致双链 DNA 结构在整体上与类似的 G：C 结构相似，但 X：K 对在局部和能量上更接近 A：T 对。
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Copyright © 2017 American Chemical Society
版权所有 © 2017 美国化学会
版权所有 © 2017 美国化学会
A quarter-century has passed since the first experiments showed that standard DNA does not exploit all hydrogen bonding patterns possible within the Watson–Crick nucleobase pairing scheme. (1) In standard pairs, the nucleobase heterocycles attached to a deoxyribose sugar through N-glycosidic bonds on antiparallel phosphodiester strands interact on a Watson–Crick edge via hydrogen bonding interactions. However, this Watson–Crick geometry can accommodate as many as 12 nucleobases forming up to as many as six orthogonal nucleobase pairs (Figure 1). (2-6) These form an artificially expanded genetic information system (AEGIS). Explorations of AEGIS have created new technology and medicine as they have opened new frontiers for the study of nucleic acids in general. (7)
自从第一次实验表明标准 DNA 并未利用 Watson-Crick 核碱基配对方案中所有可能的氢键模式以来，已经过去了四分之一个世纪。（1） 在标准对中，通过反平行磷酸二酯链上的 N-糖苷键连接到脱氧核糖的核碱基杂环通过氢键相互作用在 Watson-Crick 边缘上相互作用。然而，这种 Watson-Crick 几何形状可以容纳多达 12 个核碱基，形成多达 6 个正交核碱基对（图 1）。（2-6） 这些形成了人工扩展的遗传信息系统 （AEGIS）。对 AEGIS 的探索创造了新的技术和医学，因为它们为核酸的研究开辟了新的领域。（7）
自从第一次实验表明标准 DNA 并未利用 Watson-Crick 核碱基配对方案中所有可能的氢键模式以来，已经过去了四分之一个世纪。（1） 在标准对中，通过反平行磷酸二酯链上的 N-糖苷键连接到脱氧核糖的核碱基杂环通过氢键相互作用在 Watson-Crick 边缘上相互作用。然而，这种 Watson-Crick 几何形状可以容纳多达 12 个核碱基，形成多达 6 个正交核碱基对（图 1）。（2-6） 这些形成了人工扩展的遗传信息系统 （AEGIS）。对 AEGIS 的探索创造了新的技术和医学，因为它们为核酸的研究开辟了新的领域。（7）
Figure 1 图 1
Figure 1. Chemical structures with atom numbers for A:T, G:C, P:Z, and X:P.
图 1.具有原子数的化学结构 A：T、G：C、P：Z 和 X：P。
In particular, no standard pyrimidine nucleotide presents a hydrogen bond donor–acceptor–donor pattern to a purine that presents the complementary acceptor–donor–acceptor hydrogen bonding pattern. In AEGIS, this nonstandard pattern can be implemented by the nucleotide analogues 2,4-diamino-5-(1′-β-d-2′-deoxyribofuranosyl)-pyrimidine (implementing the pyrimidine donor–acceptor–donor hydrogen bonding pattern, K), and 8-(β-d-2′-deoxyribofuranosyl)imidazo[1,2-a]-1,3,5-triazine-2(8H)-4(3H)-dione (implementing the purine acceptor–donor–acceptor hydrogen bonding pattern, X). The first nucleobase K has a pKa of ∼6.7 (as a free species, in its protonated form. (8) The second nucleobase X has a pKa of ∼8.5, reported here for the first time based on a trace of UV as a function of pH (Supporting Information Figure S1). The X:K pair differs from natural pairs in two ways. First, a pyrimidine analogue implementing a donor–acceptor–donor hydrogen bonding pattern (like K) must be a C-glycoside, joining the heterocycle to the sugar via a carbon–carbon bond. Second, that analogue presents a hydrogen bond donor, an NH2 moiety, to the minor groove. These features of K differ in both respects from natural T and C. These are both N-glycosides, and both present a C═O unit (the oxygen being O2) to the minor groove. Further, K differs in the second respect from the AEGIS 5-nitro-1H-pyridin-2-one heterocycle (presenting the pyrimidine donor–donor–acceptor pattern, trivially Z); Z also presents a C═O unit to the minor groove. (9-12) The fact that a nearly complete molecular biology has been developed for the P:Z pair suggests that a pair with a C-glycosidic component is compatible with a synthetic biology (Figure 1).
特别是，没有标准的嘧啶核苷酸呈现氢键供体 - 受体 - 供体模式，而嘌呤则呈现互补受体 - 供体 - 受体氢键模式。在 AEGIS 中，这种非标准模式可以通过核苷酸类似物 2,4-二氨基-5-（1′-β-d-2′-脱氧呋喃核糖基）-嘧啶（实现嘧啶供体-受体-供体氢键模式，K）和 8-（β-d-2′-脱氧核糖呋喃酰基）咪唑[1,2-a]-1,3,5-三嗪-2（8H）-4（3H）-二酮（实现嘌呤受体-供体-受体氢键模式，X）来实现。第一个核碱基 K 的 pKa 为 ∼6.7（作为游离物质，以其质子化形式）。（8） 第二个核碱基 X 的 pKa 为 ∼8.5，这里首次根据痕量 UV 作为 pH 值的函数进行了报道（支持信息图 S1）。X：K 对与自然对在两个方面不同。首先，实现供体-受体-供体氢键模式的嘧啶类似物（如 K）必须是 C-糖苷，通过碳-碳键将杂环连接到糖上。其次，该类似物将氢键供体，NH2 部分，呈现到小沟中。K 的这些特征在两个方面都与自然 T 和 C 不同。这些都是 N-糖苷，并且都在小沟中呈有一个 C═O 单位（氧是 O2）。此外，K 在第二个方面与 AEGIS 5-硝基-1H-吡啶-2-酮杂环不同（呈现嘧啶供体-供体-受体模式，简单地为 Z）;Z 还为小调槽提供了一个 C═O 单元。 （9-12） 已经为 P：Z 对开发了几乎完整的分子生物学这一事实表明，具有 C-糖苷成分的对与合成生物学兼容（图 1）。
特别是，没有标准的嘧啶核苷酸呈现氢键供体 - 受体 - 供体模式，而嘌呤则呈现互补受体 - 供体 - 受体氢键模式。在 AEGIS 中，这种非标准模式可以通过核苷酸类似物 2,4-二氨基-5-（1′-β-d-2′-脱氧呋喃核糖基）-嘧啶（实现嘧啶供体-受体-供体氢键模式，K）和 8-（β-d-2′-脱氧核糖呋喃酰基）咪唑[1,2-a]-1,3,5-三嗪-2（8H）-4（3H）-二酮（实现嘌呤受体-供体-受体氢键模式，X）来实现。第一个核碱基 K 的 pKa 为 ∼6.7（作为游离物质，以其质子化形式）。（8） 第二个核碱基 X 的 pKa 为 ∼8.5，这里首次根据痕量 UV 作为 pH 值的函数进行了报道（支持信息图 S1）。X：K 对与自然对在两个方面不同。首先，实现供体-受体-供体氢键模式的嘧啶类似物（如 K）必须是 C-糖苷，通过碳-碳键将杂环连接到糖上。其次，该类似物将氢键供体，NH2 部分，呈现到小沟中。K 的这些特征在两个方面都与自然 T 和 C 不同。这些都是 N-糖苷，并且都在小沟中呈有一个 C═O 单位（氧是 O2）。此外，K 在第二个方面与 AEGIS 5-硝基-1H-吡啶-2-酮杂环不同（呈现嘧啶供体-供体-受体模式，简单地为 Z）;Z 还为小调槽提供了一个 C═O 单元。 （9-12） 已经为 P：Z 对开发了几乎完整的分子生物学这一事实表明，具有 C-糖苷成分的对与合成生物学兼容（图 1）。
As noted in previously published work, this particular X:K AEGIS pair is of interest because it has no tautomeric forms, protonated states, or deprotonated states that allow either the purine or the pyrimidine to mispair with any natural nucleobase to form a pair with a Watson–Crick geometry, that is, edge-on hydrogen bonded pair. (13, 14) Thus, the fidelity of X:K replication depends only on interactions made directly by the DNA polymerase involved. Native E. coli DNA polymerase I makes little full-length product for a template containing a single K in the absence of dXTP, but incorporates dTTP opposite X in the absence of dKTP in primer extension assays. (14) This mismatch arises through the formation of a type 1 X:T wobble structure. (13) Further, when challenged with a substrate containing two consecutive X:K pairs, DNA polymerase I has difficulty extending the primer; this is true whether the template includes KK or a mixture of K and X. (14)
正如以前发表的工作中所指出的，这个特殊的 X：K AEGIS 对很感兴趣，因为它没有互变异构形式、质子化状态或去质子化状态，这些状态允许嘌呤或嘧啶与任何天然核碱基错配，形成具有 Watson-Crick 几何形状的对，即边上氢键对。（13， 14）因此，X：K 复制的保真度仅取决于所涉及的 DNA 聚合酶直接进行的相互作用。天然大肠杆菌 DNA 聚合酶 I 在不存在 dXTP 的情况下为含有单个 K 的模板产生很少的全长产物，但在引物延伸测定中，在没有 dKTP 的情况下掺入与 X 相反的 dTTP。（14） 这种错配是通过形成 1 型 X：T 摆动结构引起的。（13） 此外，当用含有两个连续 X：K 对的底物攻击时，DNA 聚合酶 I 难以延伸引物;无论模板包含 KK 还是 K 和 X 的混合，都是如此。（14）
正如以前发表的工作中所指出的，这个特殊的 X：K AEGIS 对很感兴趣，因为它没有互变异构形式、质子化状态或去质子化状态，这些状态允许嘌呤或嘧啶与任何天然核碱基错配，形成具有 Watson-Crick 几何形状的对，即边上氢键对。（13， 14）因此，X：K 复制的保真度仅取决于所涉及的 DNA 聚合酶直接进行的相互作用。天然大肠杆菌 DNA 聚合酶 I 在不存在 dXTP 的情况下为含有单个 K 的模板产生很少的全长产物，但在引物延伸测定中，在没有 dKTP 的情况下掺入与 X 相反的 dTTP。（14） 这种错配是通过形成 1 型 X：T 摆动结构引起的。（13） 此外，当用含有两个连续 X：K 对的底物攻击时，DNA 聚合酶 I 难以延伸引物;无论模板包含 KK 还是 K 和 X 的混合，都是如此。（14）
In earlier work, xanthosine implemented the X hydrogen bonding pattern, and five cycles of replications were carried out using an HIV-1 reverse transcriptase variant. (12) The fidelity per cycle was calculated by taking the fifth root of the overall extent of loss, which was determined through the generation of a restriction site as a consequence of the loss. The wobble pair that created the loss was studied using standing start primer extension, followed by analysis of the products using gel electrophoresis and band quantitation. Here, fidelity was measured in the absence of the complement. (13)
在早期的工作中，黄嘧啶实现了 X 氢键模式，并使用 HIV-1 逆转录酶变体进行了五个复制循环。（12） 每个周期的保真度是通过取总体损失程度的五次方根来计算的，这是通过作为损失的结果产生限制位点来确定的。使用静止起始引物延伸研究产生损失的摆动对，然后使用凝胶电泳和条带定量分析产物。在这里，保真度是在没有补码的情况下测量的。（13）
在早期的工作中，黄嘧啶实现了 X 氢键模式，并使用 HIV-1 逆转录酶变体进行了五个复制循环。（12） 每个周期的保真度是通过取总体损失程度的五次方根来计算的，这是通过作为损失的结果产生限制位点来确定的。使用静止起始引物延伸研究产生损失的摆动对，然后使用凝胶电泳和条带定量分析产物。在这里，保真度是在没有补码的情况下测量的。（13）
Fidelity has also been assessed by reference to the fidelity of replication of two other AEGIS pairs, the S:B pair and the Z:P pair. With these two AEGIS pairs, in contrast to the X:K pair, mismatching without geometric distortion is possible from a minor tautomer form or a minor deprotonated form. Of course, the exact fidelity depends on the exact polymerase used, the exact molecule implementing the X hydrogen bonding pattern, and the exact conditions where the fidelity is examined.
保真度也通过参考另外两个 AEGIS 对（S：B 对和 Z：P 对）的复制保真度来评估。与 X：K 对相比，这两个 AEGIS 对可以从次要互变异构体形式或次要去质子化形式进行不匹配而不会产生几何变形。当然，确切的保真度取决于所使用的确切聚合酶、实现 X 氢键模式的确切分子以及检查保真度的确切条件。
保真度也通过参考另外两个 AEGIS 对（S：B 对和 Z：P 对）的复制保真度来评估。与 X：K 对相比，这两个 AEGIS 对可以从次要互变异构体形式或次要去质子化形式进行不匹配而不会产生几何变形。当然，确切的保真度取决于所使用的确切聚合酶、实现 X 氢键模式的确切分子以及检查保真度的确切条件。
The ability of E. coli to rescue selectable markers by mismatching was examined in living cells, which ensures fidelity of DNA replication. (14) Here, the S:B pair and the Z:P pairs, absent their complementary triphosphates, behave in large part as expected based on their tautomeric forms (for the first) and deprotonated forms (for the second). (15) The X:K pair is “seen” by the mismatch repair enzyme MutS to be a legitimate match, meaning that this DNA repair system will not excise X:K pairs in living bacterial cells. This makes the X:K pair a prime candidate to enter living systems as a fifth and sixth DNA pair, one that does not suffer from geometric distortion displayed by purely size-complementary pairs. (16, 17)
在活细胞中检查了大肠杆菌通过错配拯救选择标记物的能力，这确保了 DNA 复制的保真度。（14） 在这里，S：B 对和 Z：P 对，没有它们的互补三磷酸盐，根据它们的互变异构形式（对于第一个）和去质子化形式（对于第二个）在很大程度上表现为预期的。（15） 错配修复酶 MutS “看到”X：K 对是合法匹配的，这意味着该 DNA 修复系统不会切除活细菌细胞中的 X：K 对。这使得 X：K 对成为作为第五和第六个 DNA 对进入生命系统的主要候选者，该 DNA 对不会受到纯粹大小互补对所显示的几何失真的影响。（16， 17）
在活细胞中检查了大肠杆菌通过错配拯救选择标记物的能力，这确保了 DNA 复制的保真度。（14） 在这里，S：B 对和 Z：P 对，没有它们的互补三磷酸盐，根据它们的互变异构形式（对于第一个）和去质子化形式（对于第二个）在很大程度上表现为预期的。（15） 错配修复酶 MutS “看到”X：K 对是合法匹配的，这意味着该 DNA 修复系统不会切除活细菌细胞中的 X：K 对。这使得 X：K 对成为作为第五和第六个 DNA 对进入生命系统的主要候选者，该 DNA 对不会受到纯粹大小互补对所显示的几何失真的影响。（16， 17）
To advance in a corresponding way the synthetic biology of GACTKX DNA, we began by recognizing that nucleobases play an important role in dictating the overall structure and properties of a DNA duplex. (18) In general, duplex structures and their overall local and helical parameters play an important role in recognition of the DNA in processes such as protein binding, replication, gene regulation, and subsequent transcriptional events. Exploration of the structure of duplex DNA containing unnatural nucleotides can expand our understanding of this relation. Studies of DNA duplexes having multiple and consecutive non-natural nucleobase pairs are likely to be more informative than studies with duplexes containing only one. This was the case for Z and its partner P (7-amino-9-(1′-beta-d-2′-deoxyribofuranosyl)-imidazo[1,2-c]pyrimidin-5(1H)-one), where both structural and dynamic studies of duplexes containing multiple and consecutive Z:P pairs have advanced our understanding of GACTZP DNA, as well as DNA in general. (11, 19)
为了以相应的方式推进 GACTKX DNA 的合成生物学，我们首先认识到核碱基在决定 DNA 双链体的整体结构和特性方面起着重要作用。（18） 一般来说，双链结构及其整体局部和螺旋参数在蛋白质结合、复制、基因调控和随后的转录事件等过程中识别 DNA 起着重要作用。探索含有非天然核苷酸的双链 DNA 的结构可以扩展我们对这种关系的理解。对具有多个连续非天然核碱基对的 DNA 双链体的研究可能比对仅包含一个核碱基对的双链体的研究提供更多信息。Z 及其伴侣 P （7-氨基-9-（1′-β-d-2′-脱氧呋喃核糖基）-咪唑[1,2-c]嘧啶-5（1H）-酮）就是这种情况，其中包含多个和连续 Z：P 对的双链体的结构和动力学研究都促进了我们对 GACTZP DNA 以及一般 DNA 的理解。（11， 19）
为了以相应的方式推进 GACTKX DNA 的合成生物学，我们首先认识到核碱基在决定 DNA 双链体的整体结构和特性方面起着重要作用。（18） 一般来说，双链结构及其整体局部和螺旋参数在蛋白质结合、复制、基因调控和随后的转录事件等过程中识别 DNA 起着重要作用。探索含有非天然核苷酸的双链 DNA 的结构可以扩展我们对这种关系的理解。对具有多个连续非天然核碱基对的 DNA 双链体的研究可能比对仅包含一个核碱基对的双链体的研究提供更多信息。Z 及其伴侣 P （7-氨基-9-（1′-β-d-2′-脱氧呋喃核糖基）-咪唑[1,2-c]嘧啶-5（1H）-酮）就是这种情况，其中包含多个和连续 Z：P 对的双链体的结构和动力学研究都促进了我们对 GACTZP DNA 以及一般 DNA 的理解。（11， 19）
With the goal of understanding how multiple X:K pairs might affect the structure of duplex DNA, which is of particular interest given that E. coli DNA polymerase I has difficulty incorporating two consecutive X:K pairs, we first examined the circular dichroism (CD) spectra of this system in aqueous solution. Then, we used a host–guest system to crystallize 5′-CTTATXXTAKKATAAG-3′, referred to as 2X2K. In this system, the N-terminal fragment (residues 24–278) of Moloney murine leukemia virus reverse transcriptase (MMLV-RT) serves as the host and a 16-mer duplex DNA oligonucleotide as the guest. (20, 21) The N-terminal fragment of MMLV-RT consists of the fingers and palm domains, and the DNA binds to a site within the fingers domain involving minor groove and backbone hydrogen bonding. (22)
为了了解多个 X：K 对如何影响双链 DNA 的结构，鉴于大肠杆菌 DNA 聚合酶 I 难以掺入两个连续的 X：K 对，这一点特别有趣，我们首先检查了该系统在水溶液中的圆二色谱 （CD） 光谱。然后，我们使用主客体系统结晶 5′-CTTATXXTAKKATAAG-3′，称为 2X2K。在该系统中，莫洛尼鼠白血病病毒逆转录酶 （MMLV-RT） 的 N 末端片段（残基 24-278）作为宿主，16 聚体双链体 DNA 寡核苷酸作为客体。（20， 21）MMLV-RT 的 N 端片段由手指和手掌结构域组成，DNA 与手指结构域内涉及小沟和骨架氢键的位点结合。（22）
为了了解多个 X：K 对如何影响双链 DNA 的结构，鉴于大肠杆菌 DNA 聚合酶 I 难以掺入两个连续的 X：K 对，这一点特别有趣，我们首先检查了该系统在水溶液中的圆二色谱 （CD） 光谱。然后，我们使用主客体系统结晶 5′-CTTATXXTAKKATAAG-3′，称为 2X2K。在该系统中，莫洛尼鼠白血病病毒逆转录酶 （MMLV-RT） 的 N 末端片段（残基 24-278）作为宿主，16 聚体双链体 DNA 寡核苷酸作为客体。（20， 21）MMLV-RT 的 N 端片段由手指和手掌结构域组成，DNA 与手指结构域内涉及小沟和骨架氢键的位点结合。（22）
Table 1. Data and Refinement Statistics for 2X2K
表 1.2X2K 的数据和优化统计量
表 1.2X2K 的数据和优化统计量
| a (Å) a （Å） | 55 |
| b (Å) b （Å） | 145.6 |
| c (Å) c （Å） | 46.9 |
| space group 空间组 | P21212 |
| resolution 分辨率 | 33.72–1.75 |
| total observations 总观测值 | 484673 |
| unique reflections 独特反射 | 38305 |
| completeness 完整性 | 98.4 (92.3) |
| Rmeas (%) R测量值 （%） | 4.2 (33.8) |
| Rpim (%) Rpim （%） | 1.7 (15.4) |
| I/σ 我/σ | 37.4 (4.65) |
| Refinement Statistics 细化统计 | |
| R value (%) R 值 （%） | 20.9 |
| R free (%) 无 R （%） | 23.9 |
| RMSD bonds (Å) RMSD 债券 （Å） | 0.007 |
| RMSD angles (deg) RMSD 角（度） | 1.114 |
| Number of Atoms 原子数 | |
| protein/DNA 蛋白质/DNA | 1968/324 |
| water 水 | 188 |
| Average B-Factors 平均 B 因子 | |
| protein/DNA 蛋白质/DNA | 24.13/48.36 |
| water 水 | 26.06 |
The use of the host guest system enables us to compare the structures of DNA containing unnatural nucleobases to those of natural DNA duplexes. Since the central 10 base pairs out of the 16 base pairs are free of interaction with the protein, the structures of DNA are dictated by the sequence. (20, 22-24) Moreover, different DNA sequences crystallize in the same crystal lattice; therefore, the structural comparisons are subject to the same lattice constraints. Our structural analysis of X:K pairs is supported by computational analysis of the electrostatic potential surface, dipole, and hydrogen bonding energy.
使用宿主客体系统使我们能够比较含有非天然核碱基的 DNA 的结构与天然 DNA 双链体的结构。由于 16 个碱基对中的中心 10 个碱基对与蛋白质没有相互作用，因此 DNA 的结构由序列决定。（20,22-24）此外，不同的 DNA 序列在同一个晶格中结晶;因此，结构比较受相同的晶格约束。我们对 X：K 对的结构分析得到了静电势表面、偶极子和氢键能的计算分析的支持。
使用宿主客体系统使我们能够比较含有非天然核碱基的 DNA 的结构与天然 DNA 双链体的结构。由于 16 个碱基对中的中心 10 个碱基对与蛋白质没有相互作用，因此 DNA 的结构由序列决定。（20,22-24）此外，不同的 DNA 序列在同一个晶格中结晶;因此，结构比较受相同的晶格约束。我们对 X：K 对的结构分析得到了静电势表面、偶极子和氢键能的计算分析的支持。
Results and Discussion 结果与讨论
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Solution Properties of GATCKX
GATCKX 的解特性
The CD spectra of all four sequences (AT, GC, 2X2K, and 3K3X) exhibit B-like properties (Figure 2), with subtle differences in the peak position and heights pertaining to differences in the primary sequence of the DNA. The spectra for the AT-rich sequence exhibits a negative peak around 248 nm and a positive, longer wavelength peak at about 279 nm, typical of right-handed B-DNA. The GC-rich sequence on the other hand has a broad negative peak at around 245 nm and a positive peak for the GC-rich sequence shifted to 270 nm instead of 279 observed for the AT-rich sequence, still indicative of right-handed B-form DNA. Both 2X2K and 3K3X sequences exhibit spectra that are more similar to the GC-rich than AT-rich sequence with their broad negative peak at 250 nm and the positive peak at 273 nm. This finding suggests that the overall helical forms and the X:K chromophores more closely resemble B-form GC-rich DNA than AT-rich DNA.
所有四个序列（AT、GC、2X2K 和 3K3X）的 CD 谱图都表现出类似 B 的特性（图 2），峰位置和峰高的细微差异与 DNA 一级序列的差异有关。富含 AT 的序列的光谱在 248 nm 附近表现出一个负峰，在大约 279 nm 处显示出一个正的、波长更长的峰，这是右旋 B-DNA 的典型特征。另一方面，富含 GC 的序列在 245 nm 左右有一个宽的负峰，而富含 GC 的序列的正峰移动到 270 nm，而不是在富含 AT 的序列中观察到的 279 nm，这仍然表明右旋 B 型 DNA。2X2K 和 3K3X 序列的谱图与富含 GC 的序列相比，其谱图更类似于富含 GC 的序列，在 250 nm 处具有较宽的负峰，在 273 nm 处具有较宽的正峰。这一发现表明，整体螺旋形式和 X：K 发色团更类似于富含 B 型 GC 的 DNA，而不是富含 AT 的 DNA。
所有四个序列（AT、GC、2X2K 和 3K3X）的 CD 谱图都表现出类似 B 的特性（图 2），峰位置和峰高的细微差异与 DNA 一级序列的差异有关。富含 AT 的序列的光谱在 248 nm 附近表现出一个负峰，在大约 279 nm 处显示出一个正的、波长更长的峰，这是右旋 B-DNA 的典型特征。另一方面，富含 GC 的序列在 245 nm 左右有一个宽的负峰，而富含 GC 的序列的正峰移动到 270 nm，而不是在富含 AT 的序列中观察到的 279 nm，这仍然表明右旋 B 型 DNA。2X2K 和 3K3X 序列的谱图与富含 GC 的序列相比，其谱图更类似于富含 GC 的序列，在 250 nm 处具有较宽的负峰，在 273 nm 处具有较宽的正峰。这一发现表明，整体螺旋形式和 X：K 发色团更类似于富含 B 型 GC 的 DNA，而不是富含 AT 的 DNA。
Figure 2 图 2
Figure 2. Characterization of duplex DNA including X:K pairs by circular dichroism (CD). The ellipticity is plotted versus the wavelength for 2X2K (long dash line), 3X3K (dash dot dot line) along with control sequences for GC (dotted line), and AT (solid line). All of the duplexes have CD spectra indicative of right-handed B-form DNA.
图 2.通过环二色谱 （CD） 表征双链 DNA，包括 X：K 对。绘制了 2X2K（长虚线）、3X3K（虚线点线）的椭圆度与波长的关系图，以及 GC（虚线）和 AT（实线）的控制序列。所有双链体都有指示右旋 B 型 DNA 的 CD 光谱。
Structure of the Host–Guest Complex
主客复合体的结构
To obtain a detailed understanding of the XK-containing DNA, the self-complementary 2X2K 16-mer oligonucleotide was crystallized as a host–guest complex. The host in this system is the N-terminal fragment of Moloney murine leukemia virus reverse transcriptase. Guest DNA molecules including 2X2K that crystallize in this system are B-form in the host–guest complex. 3X3K, like the corresponding oligonucleotide containing P:Z pairs (3/6ZP), did not crystallize as a host–guest complex. DNA-only crystals of 3X3K did not diffract to high resolution in contrast to those obtained for 3/6ZP. (11) The asymmetric unit of the 2X2K crystal includes one protein molecule and half of the self-complementary 16-mer DNA oligonucleotide or an 8-mer duplex. The host–guest complex is generated by crystallographic symmetry comprising two molecules of the host protein and a 16mer DNA duplex (Figure 3). Both dK and dX yielded well-defined electron density (Figure 3) in the structure and exhibited the expected hydrogen-bonding interactions, namely three hydrogen bonds between the edge-on nucleobases. The hydrogen bonding distances between the two X:K nucleobase pairs vary from 2.64 to 3.29 Å in the 2X2K structure (Figure 3) and will be discussed in more detail below. An advantage of using this system is the ability to directly compare the structures of sequences containing G:C, A:T, or P:Z base pairs in the equivalent positions, crystallized in the same host–guest complex and thus subject to the same lattice constraints.
为了详细了解含 XK 的 DNA，将自互补的 2X2K 16 聚体寡核苷酸结晶为主客体复合物。该系统中的宿主是 Moloney 鼠白血病病毒逆转录酶的 N 端片段。在该系统中结晶的客体 DNA 分子（包括 2X2K）在主客体复合物中是 B 型。3X3K 与含有 P：Z 对 （3/6ZP） 的相应寡核苷酸一样，未以主客体复合物的形式结晶。与 3/6ZP 相比，3X3K 的仅 DNA 晶体没有衍射到高分辨率。（11） 2X2K 晶体的不对称单元包括一个蛋白质分子和一半的自互补 16 聚体 DNA 寡核苷酸或一个 8 聚体双链体。主客体复合物由晶体对称性产生，包括两个宿主蛋白分子和一个 16 聚体 DNA 双链体（图 3）。dK 和 dX 在结构中都产生了明确的电子密度（图 3），并表现出预期的氢键相互作用，即边缘核碱基之间的三个氢键。在 2X2K 结构中，两个 X：K 核碱基对之间的氢键距离从 2.64 到 3.29 Å 不等（图 3），下面将更详细地讨论。使用该系统的一个优点是能够直接比较包含等效位置的 G：C、A：T 或 P：Z 碱基对的序列的结构，这些碱基对在相同的主客体复合物中结晶，因此受到相同的晶格约束。
为了详细了解含 XK 的 DNA，将自互补的 2X2K 16 聚体寡核苷酸结晶为主客体复合物。该系统中的宿主是 Moloney 鼠白血病病毒逆转录酶的 N 端片段。在该系统中结晶的客体 DNA 分子（包括 2X2K）在主客体复合物中是 B 型。3X3K 与含有 P：Z 对 （3/6ZP） 的相应寡核苷酸一样，未以主客体复合物的形式结晶。与 3/6ZP 相比，3X3K 的仅 DNA 晶体没有衍射到高分辨率。（11） 2X2K 晶体的不对称单元包括一个蛋白质分子和一半的自互补 16 聚体 DNA 寡核苷酸或一个 8 聚体双链体。主客体复合物由晶体对称性产生，包括两个宿主蛋白分子和一个 16 聚体 DNA 双链体（图 3）。dK 和 dX 在结构中都产生了明确的电子密度（图 3），并表现出预期的氢键相互作用，即边缘核碱基之间的三个氢键。在 2X2K 结构中，两个 X：K 核碱基对之间的氢键距离从 2.64 到 3.29 Å 不等（图 3），下面将更详细地讨论。使用该系统的一个优点是能够直接比较包含等效位置的 G：C、A：T 或 P：Z 碱基对的序列的结构，这些碱基对在相同的主客体复合物中结晶，因此受到相同的晶格约束。
Figure 3 图 3
Figure 3. Crystal structure of host–guest complex including self-complementary 16 base pair oligonucleotide. (A) The N-terminal fragment of Moloney murine leukemia virus reverse transcriptase serves as the host in the complex including two protein molecules, shown as cartoon renderings in cyan and green, and a 16-mer duplex, each strand shown as a stick rendering C, cyan or green, O, red, N, blue, and phosphorus in orange; X:K pairs are shown with C in magenta. The complex depicted is that of the host–guest complex for the oligonucleotide shown with two consecutive X:K nucleobase pairs (2X2K). Within our crystals, the asymmetric unit includes only half of the complex depicted and thus the equivalent of eight nucleobase pairs and one protein molecule, indicated by the dashed line. (B) The sequence of the 2X2K sequence and position numbering for the nucleobases within the duplex. (C) The final 2Fo–Fc electron density map is shown as gray mesh renderings contoured at 1.0 σ for the X:K pair at position 6.
图 3.主客体复合物的晶体结构，包括自互补的 16 个碱基对寡核苷酸。（A） 莫洛尼鼠白血病病毒逆转录酶的 N 端片段在复合物中充当宿主，包括两个蛋白质分子，显示为青色和绿色的卡通渲染，以及一个 16 聚体双链体，每条链显示为一根棒状，渲染 C、青色或绿色、O、红色、N、蓝色和橙色磷;X：K 对以洋红色显示 C。所描绘的复合物是寡核苷酸的主客体复合物，以两个连续的 X：K 核碱基对 （2X2K） 显示。在我们的晶体中，不对称单元仅包括所描述的复合物的一半，因此相当于 8 个核碱基对和一个蛋白质分子，如虚线所示。（B） 双链体内核碱基的 2X2K 序列序列和位置编号。（C） 最终的 2Fo-F c 电子密度图显示为 6 位 X：K 对在 1.0 σ 处等高的灰色网格渲染。
The X:K pair presents a total of three electronegative atoms in the major groove as found in the natural counterparts, with potential hydrogen-bond acceptors, N5 of K and O6 of X, and a potential hydrogen-bond donor, the N4 amino group amino group of K (Figure 4). G:C, A:T, and P:Z present potential hydrogen bond acceptors including O6 of G or P and N7 of G or A and O4 of T, while the N6 amino group of A and N4 amino group of C or Z can serve as hydrogen bond donors. Z also presents the zwitterionic nitro group in the major groove, which can serve as a hydrogen bond acceptor.
X：K 对在天然对应物中发现的大凹槽中总共有三个电负性原子，具有潜在的氢键受体，K 的 N5 和 X 的 O6，以及潜在的氢键供体，K 的 N4 氨基氨基（图 4）。G：C、A：T 和 P：Z 存在潜在的氢键受体，包括 G 或 P 的 O6 和 G 或 A 的 N7 和 T 的 O4，而 A 的 N6 氨基和 C 或 Z 的 N4 氨基可以作为氢键供体。Z 还在大沟中呈现两性离子硝基，它可以作为氢键受体。
X：K 对在天然对应物中发现的大凹槽中总共有三个电负性原子，具有潜在的氢键受体，K 的 N5 和 X 的 O6，以及潜在的氢键供体，K 的 N4 氨基氨基（图 4）。G：C、A：T 和 P：Z 存在潜在的氢键受体，包括 G 或 P 的 O6 和 G 或 A 的 N7 和 T 的 O4，而 A 的 N6 氨基和 C 或 Z 的 N4 氨基可以作为氢键供体。Z 还在大沟中呈现两性离子硝基，它可以作为氢键受体。
Figure 4 图 4
Figure 4. van der Waals sphere renderings are shown with O in red, N, blue, P, orange, and C in green for X:K, yellow for P:Z, cyan for G:C, and pink for A:T for major and minor groove presentation faces of the nucleobase pairs.
图 4.范德华球体渲染以红色表示 O，X：K 以红色表示 O，以绿色表示 P：Z，以黄色表示 P：Z，以青色表示 G：C，以粉红色表示 A：T，表示核碱基对的大沟和小沟表示面。
The presence of a hydrogen-bond acceptor, either O2 or N3, associated with the pyrimidine or purine nucleobase, respectively, within the minor groove is thought to be an essential feature in natural pairs, G:C and A:T, that is “read” by DNA polymerases to ensure proper pairing during replication. (25, 26) The term “read” refers to specific hydrogen bonding interactions formed in the active site between the DNA polymerase and the template/primer substrate. The unnatural X:K pair differs from natural pairs in lacking a hydrogen-bond acceptor in the minor groove of K, which instead has a hydrogen-bond donor, the N2 amino group, while the unnatural P:Z pair retains the N3 and O2 atoms, respectively, in the minor groove. Specifically, in the minor groove, X presents N3 and O2 hydrogen-bond acceptors, while P and G presents N3 and a hydrogen-bond donor, the N2 amino group. Thus, the hydrogen bonding capabilities of X:K are unique from those of the other nucleobase pairs. Despite this, X:K is replicated faithfully. (13, 14)
在小沟内分别与嘧啶或嘌呤核碱基相关的氢键受体 O2 或 N3 的存在被认为是自然对 G：C 和 A：T 的基本特征，DNA 聚合酶“读取”以确保复制过程中的正确配对。（25， 26）术语“read”是指在 DNA 聚合酶和模板/引物底物之间的活性位点形成的特异性氢键相互作用。非自然的 X：K 对与天然对的不同之处在于，在 K 的小沟中没有氢键受体，而是有一个氢键供体，即 N2 氨基，而非自然的 P：Z 对分别在小沟中保留了 N3 和 O2 原子。具体来说，在小沟中，X 呈 N3 和 O2 氢键受体，而 P 和 G 呈 N3 和氢键供体 N2 氨基。因此，X：K 的氢键能力与其他核碱基对的氢键能力不同。尽管如此，X：K 还是被忠实地复制了。（13， 14）
在小沟内分别与嘧啶或嘌呤核碱基相关的氢键受体 O2 或 N3 的存在被认为是自然对 G：C 和 A：T 的基本特征，DNA 聚合酶“读取”以确保复制过程中的正确配对。（25， 26）术语“read”是指在 DNA 聚合酶和模板/引物底物之间的活性位点形成的特异性氢键相互作用。非自然的 X：K 对与天然对的不同之处在于，在 K 的小沟中没有氢键受体，而是有一个氢键供体，即 N2 氨基，而非自然的 P：Z 对分别在小沟中保留了 N3 和 O2 原子。具体来说，在小沟中，X 呈 N3 和 O2 氢键受体，而 P 和 G 呈 N3 和氢键供体 N2 氨基。因此，X：K 的氢键能力与其他核碱基对的氢键能力不同。尽管如此，X：K 还是被忠实地复制了。（13， 14）
Helical Properties of Duplex DNA Containing X:K Pairs
含有 x：K 对的双链 DNA 的螺旋特性
The helical parameters of the 2X2K structure were analyzed using 3DNA, which uses El Hassan and Calladine’s method (27) to calculate the major and minor groove widths as well as other base pair parameters, (28-30) and compared to other parameters of the host–guest structures of B-form DNA in which X:K is replaced with G:C, A:T, and P:Z at the same positions, PDB IDs 4XPE, 4XPC, and 4XO0, respectively. (11) The sequences used for comparison are listed in Table 2. Overall, the 2X2K structure maintains the B-helical form with an average helical twist of 34.9° ± 4.18°, corresponding to 10.3 base pairs per turn. In comparison to the 2X2K structure, GC, AT, and PZ structures have an average helical twist of 34.7° corresponding to 10.4 base pairs per turn. Thus, incorporation of two consecutive X:K pairs in the DNA sequence does not perturb the overall helical form of the DNA duplex significantly. Variations in helical twist of the X:K base pair steps are similar to those observed in all of the structures for the same base pair positions.
使用 3DNA 分析 2X2K 结构的螺旋参数，该 3DNA 使用 El Hassan 和 Calladine 的方法 （27） 计算主要和次要沟宽以及其他碱基对参数 （28-30），并与 B 型 DNA 的主客体结构的其他参数进行比较，其中 X：K 被 G 取代：C、A：T 和 P：Z 位于相同位置，PDB ID 分别为 4XPE、4XPC 和 4XO0。（11） 用于比较的序列列于表 2 中。总体而言，2X2K 结构保持了 B 螺旋形式，平均螺旋扭曲为 34.9° ± 4.18°，相当于每圈 10.3 个碱基对。与 2X2K 结构相比，GC、AT 和 PZ 结构的平均螺旋扭曲为 34.7°，相当于每圈 10.4 个碱基对。因此，在 DNA 序列中掺入两个连续的 X：K 对不会显着扰乱 DNA 双链体的整体螺旋形式。X：K 碱基对步骤的螺旋扭曲变化与在相同碱基对位置的所有结构中观察到的变化相似。
使用 3DNA 分析 2X2K 结构的螺旋参数，该 3DNA 使用 El Hassan 和 Calladine 的方法 （27） 计算主要和次要沟宽以及其他碱基对参数 （28-30），并与 B 型 DNA 的主客体结构的其他参数进行比较，其中 X：K 被 G 取代：C、A：T 和 P：Z 位于相同位置，PDB ID 分别为 4XPE、4XPC 和 4XO0。（11） 用于比较的序列列于表 2 中。总体而言，2X2K 结构保持了 B 螺旋形式，平均螺旋扭曲为 34.9° ± 4.18°，相当于每圈 10.3 个碱基对。与 2X2K 结构相比，GC、AT 和 PZ 结构的平均螺旋扭曲为 34.7°，相当于每圈 10.4 个碱基对。因此，在 DNA 序列中掺入两个连续的 X：K 对不会显着扰乱 DNA 双链体的整体螺旋形式。X：K 碱基对步骤的螺旋扭曲变化与在相同碱基对位置的所有结构中观察到的变化相似。
Table 2. Oligonucleotides Used for Analysis
表 2.用于分析的寡核苷酸
表 2.用于分析的寡核苷酸
All four structures including 2X2K, PZ, GC, and AT are B-form throughout, but exhibit some differences in minor and major groove widths. Overall, the major and minor groove widths for 2X2K resemble those of GC and PZ (Figure 5 and Table 3). The average minor and major groove widths of 12.3 and 18.3 Å, respectively, associated with the dinucleotide steps containing X:K pairs are similar to those of the corresponding dinucleotide steps in the GC structure of 12.4 and 18.0 Å and PZ structure of 12.5 and 18.7 Å, respectively (Table 3). AT-rich sequences have a narrower minor groove, as observed in our AT structure with an average minor groove width of 9.7 Å. The minor groove of 2X2K is wider by 2.6 Å than that of the AT structure. On the other hand, the major groove of 2X2K is narrower by 0.8 Å than the AT structure with major groove width of 19.1 Å.
包括 2X2K、PZ、GC 和 AT 在内的所有四种结构都呈 B 型，但在小槽宽和大槽宽度上表现出一些差异。总体而言，2X2K 的大槽宽和短槽宽度类似于 GC 和 PZ 的宽（图 5 和表 3）。与含有 X：K 对的二核苷酸步骤相关的平均短沟宽和大沟宽分别为 12.3 Å 和 18.3 Å，这与 GC 结构中相应的二核苷酸阶限分别为 12.4 和 18.0 Å 以及 PZ 结构 12.5 和 18.7 Å 相似（表 3）。富含 AT 的序列具有较窄的小沟，正如我们的 AT 结构中观察到的那样，平均小沟宽度为 9.7 Å。2X2K 的小沟比 AT 结构的小沟宽 2.6 Å。另一方面，2X2K 的大槽比大槽宽度为 19.1 Å 的 AT 结构窄 0.8 Å。
包括 2X2K、PZ、GC 和 AT 在内的所有四种结构都呈 B 型，但在小槽宽和大槽宽度上表现出一些差异。总体而言，2X2K 的大槽宽和短槽宽度类似于 GC 和 PZ 的宽（图 5 和表 3）。与含有 X：K 对的二核苷酸步骤相关的平均短沟宽和大沟宽分别为 12.3 Å 和 18.3 Å，这与 GC 结构中相应的二核苷酸阶限分别为 12.4 和 18.0 Å 以及 PZ 结构 12.5 和 18.7 Å 相似（表 3）。富含 AT 的序列具有较窄的小沟，正如我们的 AT 结构中观察到的那样，平均小沟宽度为 9.7 Å。2X2K 的小沟比 AT 结构的小沟宽 2.6 Å。另一方面，2X2K 的大槽比大槽宽度为 19.1 Å 的 AT 结构窄 0.8 Å。
Figure 5 图 5
Figure 5. Comparison of helical properties for host–guest complexes including X:K, G:C, A:T, and P:Z. The unique 8-mer DNA structure including X:K pairs (green) is shown superimposed in (A) with A:T (gray), (B) with G:C (blue), and (C) with P:Z (yellow). (D) The associated minor groove widths for the 16-mer DNA structures are shown in the same colors as designated in stuctures A–C.
图 5.主客体复合物（包括 X：K、G：C、A：T 和 P：Z）的螺旋特性比较。独特的 8 聚体 DNA 结构（包括 X：K 对（绿色）叠加在 （A） 与 A：T（灰色）、（B） 与 G：C（蓝色）和 （C） 与 P：Z（黄色）中叠加。（D） 16 聚体 DNA 结构的相关小沟宽度以与结构 A-C 中指定的颜色相同的颜色显示。
Table 3. Helical Parameters and Local Base Pair Parameters: AT, GC, 2X2K, and PZ
表 3.螺旋参数和局部碱基对参数：AT、GC、2X2K 和 PZ
表 3.螺旋参数和局部碱基对参数：AT、GC、2X2K 和 PZ
| local base pair parameters at position 6 位置 6 的局部碱基对参数 | ||||
|---|---|---|---|---|
| X-K | P-Z | G-C | A-T | |
| shear (Å) 剪切 （Å） | –0.44 | –0.87 | –1.40 | –0.22 |
| stretch (Å) 伸展 （Å） | 0.08 | –0.42 | –0.47 | –0.34 |
| stagger (Å) 交错 （Å） | 0.23 | –0.41 | –0.02 | 0.18 |
| buckle (deg) 带扣 （DEG） | 6.19 | –11.91 | 5.38 | –1.40 |
| propeller (deg) 螺旋桨 （DEG） | –10.61 | –6.69 | –5.60 | –15.89 |
| opening (deg) 开口（度） | 6.64 | 2.96 | 12.60 | 8.06 |
| local base pair parameters at position 7 位置 7 的局部碱基对参数 | ||||
|---|---|---|---|---|
| X-K | P-Z | G-C | A-T | |
| shear (Å) 剪切 （Å） | 0.08 | 0.84 | –0.68 | 0.39 |
| stretch (Å) 伸展 （Å） | –0.07 | –0.25 | –0.66 | –0.21 |
| stagger (Å) 交错 （Å） | 0.23 | 0.17 | 0.32 | 0.33 |
| buckle (deg) 带扣 （DEG） | –3.09 | 0.05 | –0.45 | 3.23 |
| propeller (deg) 螺旋桨 （DEG） | –19.41 | –10.05 | –12.64 | –17.58 |
| opening (deg) 开口（度） | –8.10 | 0.30 | –4.85 | 5.93 |
| Local base pair step parameters 本地碱基对步长参数 | ||||
|---|---|---|---|---|
| XX/KK | PP/ZZ | GG/CC GG/CC 系列 | AA/TT AA/TT 系列 | |
| shift (Å) 移位 （Å） | –2.03 | –0.93 | –1.62 | –0.29 |
| slide (Å) 滑轨 （Å） | 0.84 | –0.02 | 0.99 | –0.22 |
| rise (Å) 上升 （Å） | 3.43 | 3.03 | 3.49 | 3.13 |
| tilt (deg) 倾斜（度） | –5.12 | –6.26 | –5.74 | –1.17 |
| roll (deg) 卷（度） | –4.82 | 4.31 | –5.58 | –3.15 |
| twist (deg) 扭曲（度） | 39.29 | 39.03 | 43.67 | 39.74 |
| helical parameters 螺旋参数 | ||||
|---|---|---|---|---|
| XX/KK | PP/ZZ | GG/CC GG/CC 系列 | AA/TT AA/TT 系列 | |
| x-displacement (Å) x 位移 （Å） | 1.83 | –0.51 | 1.87 | 0.02 |
| y-displacement (Å) Y 轴排量 （Å） | 2.35 | 0.68 | 1.58 | 0.30 |
| helical-rise (Å) 螺旋上升 （Å） | 3.54 | 3.12 | 3.52 | 3.14 |
| inclination (deg) 倾角 （deg） | –7.10 | 6.38 | –7.43 | –4.63 |
| tip (deg) 尖端（度） | 7.53 | 9.27 | 7.64 | 1.71 |
| helical-twist (deg) 螺旋扭曲 （deg） | 39.89 | 39.74 | 44.36 | 39.88 |
| other parameters 其他参数 | ||||
|---|---|---|---|---|
| X:K X：K | P:Z P：Z | G:C G：C | A:T 答：T | |
| overall helical twist (deg) 整体螺旋扭曲 （deg） | 34.93 (4.18 SD) 34.93 （4.18 标清） | 34.69 (8.05 SD) 34.69 （8.05 标清） | 34.70 (7.19 SD) 34.70 （7.19 标清） | 34.70 (3.49 SD) 34.70 （3.49 标清） |
| averagea minor groove width (Å) 平均小沟宽度 （Å） | 12.3 | 12.5 | 12.4 | 9.7 |
| averagea major groove width (Å) 平均大槽宽度 （Å） | 18.3 | 18.7 | 18.0 | 19.1 |
a
a
Average values obtained for dinucleotide steps 5–7 containing X:K, Z:P, G:C, or A:T, respectively.
a
分别包含 X：K、Z：P、G：C 或 A：T 的二核苷酸步骤 5-7 获得的平均值。
Other helical parameters including x-displacement, y-displacement, helical rise, inclination, and tip are similar to the GC structure as shown in Table 3. The helical twist angle for the X:K base pairs also lies in the range usually reported for B form DNA, with X:K having a helical twist of 39.89° (B-form DNA has helical twist values ranging between 27.9° to 40°). (31-34)X:K pairs resemble more closely G:C pairs than P:Z pairs in terms of local helical conformations. Minor variations in the values of helical parameters between GC and 2X2K are a result of sequence-dependent effects on the structure.
其他螺旋参数（包括 x 位移、y 位移、螺旋上升、倾角和尖端）与表 3 所示的 GC 结构相似。X：K 碱基对的螺旋扭曲角也位于 B 型 DNA 通常报道的范围内，其中 X：K 的螺旋扭曲为 39.89°（B 型 DNA 的螺旋扭曲值在 27.9° 至 40° 之间）。就局部螺旋构象而言，（31-34）X：K 对比 P：Z 对更接近 G：C 对。GC 和 2X2K 之间螺旋参数值的微小变化是结构上序列依赖性影响的结果。
其他螺旋参数（包括 x 位移、y 位移、螺旋上升、倾角和尖端）与表 3 所示的 GC 结构相似。X：K 碱基对的螺旋扭曲角也位于 B 型 DNA 通常报道的范围内，其中 X：K 的螺旋扭曲为 39.89°（B 型 DNA 的螺旋扭曲值在 27.9° 至 40° 之间）。就局部螺旋构象而言，（31-34）X：K 对比 P：Z 对更接近 G：C 对。GC 和 2X2K 之间螺旋参数值的微小变化是结构上序列依赖性影响的结果。
Local Properties of the X:K Nucleobase Pair
X：K 核碱基对的局部性质
Local base pair parameters, local base pair step parameters, and groove widths for the X:K pair were analyzed using 3DNA (Table 3). In comparing the local base pair parameters for X:K with P:Z, G:C, and A:T, the buckle values for positions 6 and 7 (refer to Figure 1 for numbering scheme), 6.19° and −3.09°, respectively, are similar to G:C values, 5.38° and −0.45°, but less similar to those for P:Z, −11.91° and 0.05° (Table 3). Shear, stagger, and opening values are within the range observed for the other base pairs, while stretch values of 0.08 and −0.07 Å for X:K pairs in positions 6 and 7 are smaller in magnitude than those observed for other base pairs.
使用 3DNA 分析 X：K 对的局部碱基对参数、局部碱基对步骤参数和沟宽（表 3）。在比较 X：K 与 P：Z、G：C 和 A：T 的局部碱基对参数时，位置 6 和 7（编号方案参见图 1）的屈曲值分别为 6.19° 和 −3.09°，与 G：C 值 5.38° 和 −0.45° 相似，但与 P：Z 的 −11.91° 和 0.05° 的相似度较低（表 3).剪切、交错和开口值位于其他碱基对的观测范围内，而位置 6 和 7 的 X：K 对的 0.08 和 −0.07 Å 拉伸值在幅度上小于其他碱基对的观测值。
使用 3DNA 分析 X：K 对的局部碱基对参数、局部碱基对步骤参数和沟宽（表 3）。在比较 X：K 与 P：Z、G：C 和 A：T 的局部碱基对参数时，位置 6 和 7（编号方案参见图 1）的屈曲值分别为 6.19° 和 −3.09°，与 G：C 值 5.38° 和 −0.45° 相似，但与 P：Z 的 −11.91° 和 0.05° 的相似度较低（表 3).剪切、交错和开口值位于其他碱基对的观测范围内，而位置 6 和 7 的 X：K 对的 0.08 和 −0.07 Å 拉伸值在幅度上小于其他碱基对的观测值。
The X:K base pair is not planar, as evidenced by propeller twist angles of −10.61° and −19.41° for positions 6 and 7, respectively, closer to those observed for A:T (−15.98° and −17.58°) than G:C (−5.6° and −12.64°) or P:Z (−6.69° and −10.05°) (Figure 6A and Table 3). Significant propeller angles in A:T pairs are often attributed to the fact that there are only two hydrogen bonds in the pair. However, this feature in the X:K pair, which has three hydrogen bonds, suggests that it is perhaps an inherent property of the base pair itself and not dependent upon the number of hydrogen bonds. Stacking interactions of X:K and A:T pairs are superficially similar as shown in Figure 6B.
X：K 碱基对不是平面的，位置 6 和 7 的螺旋桨扭转角分别为 -10.61° 和 -19.41°，比 G：C（-5.6° 和 -12.64°）或 P：Z（-6.69° 和 -10.05°）更接近 A：T（-15.98° 和 -17.58°）观察到的扭曲角（图 6A 和表 3）).A：T 对中的重要螺旋桨角通常归因于该对中只有两个氢键的事实。然而，具有三个氢键的 X：K 对中的这一特征表明，它可能是碱基对本身的固有特性，不取决于氢键的数量。X：K 和 A：T 对的堆叠交互在表面上相似，如图 6 B 所示。
X：K 碱基对不是平面的，位置 6 和 7 的螺旋桨扭转角分别为 -10.61° 和 -19.41°，比 G：C（-5.6° 和 -12.64°）或 P：Z（-6.69° 和 -10.05°）更接近 A：T（-15.98° 和 -17.58°）观察到的扭曲角（图 6A 和表 3）).A：T 对中的重要螺旋桨角通常归因于该对中只有两个氢键的事实。然而，具有三个氢键的 X：K 对中的这一特征表明，它可能是碱基对本身的固有特性，不取决于氢键的数量。X：K 和 A：T 对的堆叠交互在表面上相似，如图 6 B 所示。
Figure 6 图 6
Figure 6. Comparison of local base pair properties of X:K and A:T. (A) Cartoon/stick renderings for X:K and A:T pairs (7th nucleotide step, X7:K10 as shown in Figure 3) with a semitransparent gray molecular surface superimposed show significant propeller twist angles for the base plane of X or A vs K or T. A similar rendering is shown for a relatively planar G:C base pair (6th nucleotide step G6:C11). (B) Stacking of XX/KK and AA/TT shown as stick renderings are overall similar in accommodating the nucleobase pairs with significant propeller twist angles.
图 6.X：K 和 A：T 的局部碱基对特性的比较。（a） X：K 和 A：T 对的卡通/棒状渲染（第 7 个核苷酸步骤，X7：K10，如图 3 所示）与半透明灰色分子表面叠加显示 X 或 A 与 K 或 T 的基平面有明显的螺旋桨扭曲角。对于相对平面的 G：C 碱基对（第 6 个核苷酸步骤 G6：C11），显示了类似的渲染。（B） 以棒状渲染显示的 XX/KK 和 AA/TT 的堆叠在容纳具有显着螺旋桨扭转角的核碱基对方面总体上相似。
It was therefore of interest to consider the electrostatic potential surface (ESP), dipole, and hydrogen bonding energies for X:K, A:T, G:C, and P:Z pairs. The ESP approximates the short-range electronic environment, while the dipole describes a longer-range electronic effect and the hydrogen bond energies, the tightness of the Watson–Crick pair. The ESP visually displays that there are different hydrogen bonding opportunities in X:K vs A:T (Figure 7). Within the ESP map, we have also displayed the electrical dipole moments. X:K has a very different electric dipole moment than A:T, both in magnitude and direction. The electrical dipole moment for the X:K pair is rotated ∼90 deg relative to A:T. Moreover, the A:T dipole is 2.1 D whereas the dipole in X:K is only 1.3 D. The Watson–Crick hydrogen bonding energy of A:T is greater than X:K by 3.6 kcal/mol (Table 4). Thus, the contributions from three hydrogen bonds in X:K energetically are weaker than for two hydrogen bonds in A:T. The estimate of the A:T electronic binding energy matches previous calculations done. (35) The Gibbs energy of forming the X:K pair is just barely negative at −1.9 kcal/mol. Note this only tells us what the hydrogen bonding contribution to the energy is, and previous studies have shown that base-pair stacking dispersion interactions are more numerically significant to the total Gibbs energy. (36) The Watson–Crick hydrogen bonding free energy utilized CCSD(T) (37-39) /aug″-cc-pVTZ for the electronic energy, which has been shown to be accurate to ∼1 kcal/mol of the true gas-phase energy of single-reference wave functions; entropic contribution was based on the M06-2X/aug″-cc-pVTZ partition function. The overall point is clear that X:K hydrogen bonding energy per hydrogen bond is significantly weaker than A:T, allowing the net contribution of the X:K pair toward duplex stability that is slightly less than the net contribution by A:T pair, notwithstanding it having three hydrogen bonds. These calculations are consistent with lower experimental melting temperatures determined for oligonucleotides including X:K pairs (SI, Table S1).
因此，考虑 X：K、A：T、G：C 和 P：Z 对的静电势表面 （ESP）、偶极子和氢键能是很有趣的。ESP 近似于短程电子环境，而偶极子描述长程电子效应和氢键能，即 Watson-Crick 对的紧密性。ESP 直观地显示 X：K 和 A：T 中存在不同的氢键机会（图 7）。在 ESP 映射中，我们还显示了电偶极矩。X：K 的电偶极矩与 A：T 在大小和方向上都非常不同。X：K 对的电偶极矩相对于 A：T 旋转 ∼90 度。此外，A：T 偶极子为 2.1 D，而 X：K 中的偶极子仅为 1.3 D。A：T 的 Watson-Crick 氢键能比 X：K 大 3.6 kcal/mol（表 4）。因此，X：K 中三个氢键在能量上的贡献比 A：T 中两个氢键的贡献弱。A：T 电子结合能的估计值与之前所做的计算相匹配。（35） 形成 X：K 对的吉布斯能量在 -1.9 kcal/mol 时几乎为零。请注意，这只能告诉我们氢键对能量的贡献是什么，以前的研究表明，碱基对堆叠分散相互作用在数值上对总吉布斯能量的意义更大。 （36） Watson-Crick 氢键自由能利用 CCSD（T） （37-39） /aug“-cc-pVTZ 作为电子能，这已被证明精确到单参考波函数的真实气相能量的 ∼1 kcal/mol;熵贡献基于 M06-2X/aug“-cc-pVTZ 分区函数。总体观点很清楚，每个氢键的 X：K 氢键能明显弱于 A：T，因此 X：K 对双链体稳定性的净贡献略小于 A：T 对的净贡献，尽管它有三个氢键。这些计算结果与为包括 X：K 对在内的寡核苷酸测定的较低实验熔解温度一致（SI，表 S1）。
因此，考虑 X：K、A：T、G：C 和 P：Z 对的静电势表面 （ESP）、偶极子和氢键能是很有趣的。ESP 近似于短程电子环境，而偶极子描述长程电子效应和氢键能，即 Watson-Crick 对的紧密性。ESP 直观地显示 X：K 和 A：T 中存在不同的氢键机会（图 7）。在 ESP 映射中，我们还显示了电偶极矩。X：K 的电偶极矩与 A：T 在大小和方向上都非常不同。X：K 对的电偶极矩相对于 A：T 旋转 ∼90 度。此外，A：T 偶极子为 2.1 D，而 X：K 中的偶极子仅为 1.3 D。A：T 的 Watson-Crick 氢键能比 X：K 大 3.6 kcal/mol（表 4）。因此，X：K 中三个氢键在能量上的贡献比 A：T 中两个氢键的贡献弱。A：T 电子结合能的估计值与之前所做的计算相匹配。（35） 形成 X：K 对的吉布斯能量在 -1.9 kcal/mol 时几乎为零。请注意，这只能告诉我们氢键对能量的贡献是什么，以前的研究表明，碱基对堆叠分散相互作用在数值上对总吉布斯能量的意义更大。 （36） Watson-Crick 氢键自由能利用 CCSD（T） （37-39） /aug“-cc-pVTZ 作为电子能，这已被证明精确到单参考波函数的真实气相能量的 ∼1 kcal/mol;熵贡献基于 M06-2X/aug“-cc-pVTZ 分区函数。总体观点很清楚，每个氢键的 X：K 氢键能明显弱于 A：T，因此 X：K 对双链体稳定性的净贡献略小于 A：T 对的净贡献，尽管它有三个氢键。这些计算结果与为包括 X：K 对在内的寡核苷酸测定的较低实验熔解温度一致（SI，表 S1）。
Figure 7 图 7
Figure 7. Dipole moments and electrostatic potential maps (ESPs) for (A) X:K, (B) A:T, (C) G:C, (D) P:Z nucleobase pairs. Methyl substituents are used in place of the C1′ carbon in the deoxyribose ring. The ESP color gradation is such that red is ca. −40 kcal/mol and blue is ca. +40 kcal/mol; strict energetic interpretation is limited to indications of broad differences in reactivity. Dipole moments are shown as a blue arrow, following the convention that a positive vector points toward a positive charge density. The magnitude of the vectors is not proportional to length (length modified for ease of view).
图 7.（A） X：K、（B） A：T、（C） G：C、（D） P：Z 核碱基对的偶极矩和静电势图 （ESP）。使用甲基取代基代替脱氧核糖环中的 C1' 碳。ESP 色阶是这样的，红色约为 −40 kcal/mol，蓝色约为 +40 kcal/mol;严格的能量解释仅限于反应性差异的广泛指征。偶极矩显示为蓝色箭头，遵循正矢量指向正电荷密度的约定。矢量的大小与长度不成比例（为便于查看，修改了长度）。
Table 4. Watson–Crick Pair Hydrogen Bonding Energy Contributions (kcal/mol) for X:K and A:T Nucleobase Pairs
表 4.X：K 和 A：T 核碱基对的 Watson-Crick 氢键对氢键能量贡献 （kcal/mol）
表 4.X：K 和 A：T 核碱基对的 Watson-Crick 氢键对氢键能量贡献 （kcal/mol）
| X:K X：K | A:T 答：T | Δ | |
|---|---|---|---|
| ΔU (electronic) ΔU （电子式） | –15.2 | –16.1 | +0.9 |
| ΔH | –13.8 | –13.1 | –0.7 |
| –TΔS | 11.9 | 7.6 | +4.3 |
| ΔG | –1.9 | –5.5 | +3.6 |
Of note, in the calculated structures of the X:K pair, the proton on N1 of X is apparently transferred to N3 of K. This transfer appears to produce the most favorable arrangement of secondary interactions proposed by Jorgensen and Pranata. (40) In their analysis, a symmetric pattern of partial positive and negative charges in forming three hydrogen bonds is the least favorable arrangement. This is the hydrogen bonding arrangement depicted for X:K in Figure 1. Alternatively, deprotonation of N1 in X and protonation of N3 in K produces the most favorable secondary arrangement with three partial positive charges associated with K and three partial negative charges associated with X as depicted in Figure 8 along with the calculated electron density supporting this arrangement for the protons. Alternatively, one could say that the energy of the endergonic acid–base transfer (given the pKa values of ∼6.7 for K8 and pKa of 8.5 for X, Figure S1) comes at the expense of the exergonic hydrogen bonds formed. This helps us to understand why three hydrogen bonds are not better than two: we only achieved this at the expense of a proton transfer.
值得注意的是，在 X：K 对的计算结构中，X 的 N1 上的质子显然转移到了 K 的 N3 上。这种转移似乎产生了 Jorgensen 和 Pranata 提出的最有利的次级相互作用安排。（40） 在他们的分析中，部分正电荷和负电荷形成三个氢键的对称模式是最不利的安排。这是图 1 中 X：K 描述的氢键排列。或者，X 中 N1 的去质子化和 K 中 N3 的质子化产生最有利的二次排列，如图 8 所示，三个部分正电荷与 K 相关，三个部分负电荷与 X 相关，以及计算出的电子密度支持质子的这种排列。或者，可以说内能酸-碱转移的能量（假设 K8 的 pKa 值为 ∼6.7，X 的 pKa 值为 8.5，图 S1）是以牺牲形成的放能氢键为代价的。这有助于我们理解为什么三个氢键不比两个好：我们只是以牺牲质子转移为代价来实现这一点。
值得注意的是，在 X：K 对的计算结构中，X 的 N1 上的质子显然转移到了 K 的 N3 上。这种转移似乎产生了 Jorgensen 和 Pranata 提出的最有利的次级相互作用安排。（40） 在他们的分析中，部分正电荷和负电荷形成三个氢键的对称模式是最不利的安排。这是图 1 中 X：K 描述的氢键排列。或者，X 中 N1 的去质子化和 K 中 N3 的质子化产生最有利的二次排列，如图 8 所示，三个部分正电荷与 K 相关，三个部分负电荷与 X 相关，以及计算出的电子密度支持质子的这种排列。或者，可以说内能酸-碱转移的能量（假设 K8 的 pKa 值为 ∼6.7，X 的 pKa 值为 8.5，图 S1）是以牺牲形成的放能氢键为代价的。这有助于我们理解为什么三个氢键不比两个好：我们只是以牺牲质子转移为代价来实现这一点。
Figure 8 图 8
Figure 8. Calculated X:K pair has a proton transfer. (A) The chemical structure is shown for the calculated X:K pair with bond distances in the vicinity of the hydrogen bonding pairs. Bond lengths (Å) of key species for Watson–Crick binding of X:K. All calculations are based on M06-2X/aug″-cc-pVDZ geometries, expected to be accurate to within 0.03 Å. Note the proton transfer from X to K, as shown by the bond lengths. (B) The calculated electron density is shown for the X:K pair and clearly shows that the proton involved in the central hydrogen bonding pair is associated with N3 of K and not N1 of X. Electron density contours given at 0.04 electron charge/Bohr3 from the KS-DFT density matrix.
图 8.计算出的 X：K 对具有质子转移。（A） 显示了计算的 X：K 对的化学结构，键距离在氢键对附近。X：K 的 Watson-Crick 结合关键物质的键长 （Å）。所有计算均基于 M06-2X/aug“-cc-pVDZ 几何结构，预计精度在 0.03 Å 以内。请注意从 X 到 K 的质子转移，如键长所示。（B） 计算出的电子密度显示了 X：K 对，并清楚地表明参与中心氢键对的质子与 K 的 N3 相关，而不是 X 的 N1。KS-DFT 密度矩阵中给出的电子密度等值线为 0.04 个电子电荷/玻尔3。
One might be skeptical of the computational claim that an acid–base transfer is necessary to form the Watson–Crick pair. This finding is independent of the gas-phase DFT calculation, however. If one uses an implicit solvent model (Cramer and Truhlar’s SMD model SMD) to represent an aqueous solvent with the same DFT protocol, the proton transfer is also observed (1.05 Å hydrogen-bond distance to the K nitrogen, 1.79 Å to the X nitrogen). Similarly, if one uses an ab initio wave function calculation with no empirical parameters such as MP2 (otherwise known as MBPT(2)), the same features are observed (1.09 Å to the K nitrogen, 1.62 Å to the X nitrogen).
人们可能会对计算声明持怀疑态度，即酸碱转移是形成 Watson-Crick 对所必需的。然而，这一发现与气相 DFT 计算无关。如果使用隐式溶剂模型（Cramer 和 Truhlar 的 SMD 模型 SMD）来表示具有相同 DFT 方案的水性溶剂，则还会观察到质子转移（与 K 氮的氢键距离为 1.05 Å，与 X 氮的氢键距离为 1.79 Å）。同样，如果使用没有经验参数（如 MP2（也称为 MBPT（2）的从头计算，则会观察到相同的特征（1.09 Å 到 K 氮，1.62 Å 到 X 氮）。
人们可能会对计算声明持怀疑态度，即酸碱转移是形成 Watson-Crick 对所必需的。然而，这一发现与气相 DFT 计算无关。如果使用隐式溶剂模型（Cramer 和 Truhlar 的 SMD 模型 SMD）来表示具有相同 DFT 方案的水性溶剂，则还会观察到质子转移（与 K 氮的氢键距离为 1.05 Å，与 X 氮的氢键距离为 1.79 Å）。同样，如果使用没有经验参数（如 MP2（也称为 MBPT（2）的从头计算，则会观察到相同的特征（1.09 Å 到 K 氮，1.62 Å 到 X 氮）。
The fact that the hydrogen bonding is weaker in X:K than in A:T likely contributes to the observed propensity to propeller. As noted above, the X:K pairs each include one hydrogen bond longer than 3.0 Å. For position 6, O2–N2, N1–N3, and O6–N4 hydrogen bonding distances are 2.64, 2.98, and 3.18 Å, respectively; for position 7, O2–N2, N1–N3, and O6–N4 are 3.28, 2.98, and 2.69 Å, respectively (see Figure 1 for atom numbers, Figure 3 for position numbers). Thus, for position 6, the long hydrogen bond is the O6–N4 bond, while for position 7, it is the O2–N2 bond. This finding is consistent with weaker theoretical hydrogen bonding in general, larger propeller angles, and potentially a contribution from buckling as well. The X:K pair at position 6 has a buckle angle of 6.2° and propeller angle of −10.6°, while position 7 buckles in the opposite direction, buckle angle of −3.1°, and has a propeller angle of −19.4°. Similarly, the A:T pairs have one long hydrogen bond for positions 6 and 7 (numbering shown in Figure 3), in this case the same bond, N6–O4, 3.08, and 3.13 Å, respectively. Both of the A:T pairs have large propeller angles, −15.9 and −17.6° with small buckle angles, −1.4° and 3.2°, respectively.
X：K 中的氢键比 A：T 中的氢键弱这一事实可能有助于观察到螺旋桨的倾向。如上所述，每个 X：K 对都包含一个长度超过 3.0 Å 的氢键。对于位置 6，O2-N2、N1-N3 和 O6-N4 氢键距离分别为 2.64、2.98 和 3.18 Å;对于位置 7，O2–N2、N1–N3 和 O6–N4 分别为 3.28、2.98 和 2.69 Å（原子数见图 1，位置数见图 3）。因此，对于位置 6，长氢键是 O6-N4 键，而对于位置 7，它是 O2-N2 键。这一发现与一般较弱的理论氢键、较大的螺旋桨角度以及屈曲可能也有助于的结果一致。位置 6 的 X：K 对的弯曲角为 6.2°，螺旋桨角为 −10.6°，而位置 7 的弯曲方向相反，弯曲角为 -3.1°，螺旋桨角为 −19.4°。同样，A：T 对在位置 6 和 7 有一个长氢键（编号如图 3 所示），在本例中是相同的键，分别为 N6-O4、3.08 和 3.13 Å。A：T 对都有大螺旋桨角，分别为 -15.9 和 -17.6°，带小扣角，分别为 -1.4° 和 3.2°。
X：K 中的氢键比 A：T 中的氢键弱这一事实可能有助于观察到螺旋桨的倾向。如上所述，每个 X：K 对都包含一个长度超过 3.0 Å 的氢键。对于位置 6，O2-N2、N1-N3 和 O6-N4 氢键距离分别为 2.64、2.98 和 3.18 Å;对于位置 7，O2–N2、N1–N3 和 O6–N4 分别为 3.28、2.98 和 2.69 Å（原子数见图 1，位置数见图 3）。因此，对于位置 6，长氢键是 O6-N4 键，而对于位置 7，它是 O2-N2 键。这一发现与一般较弱的理论氢键、较大的螺旋桨角度以及屈曲可能也有助于的结果一致。位置 6 的 X：K 对的弯曲角为 6.2°，螺旋桨角为 −10.6°，而位置 7 的弯曲方向相反，弯曲角为 -3.1°，螺旋桨角为 −19.4°。同样，A：T 对在位置 6 和 7 有一个长氢键（编号如图 3 所示），在本例中是相同的键，分别为 N6-O4、3.08 和 3.13 Å。A：T 对都有大螺旋桨角，分别为 -15.9 和 -17.6°，带小扣角，分别为 -1.4° 和 3.2°。
On the other hand, P:Z hydrogen bonding distances at position 6 for N2–O2, N1–N3, and O6–N4 are 2.57, 2.79, and 2.94 Å, respectively, and the values for position 7 P:Z are 2.63, 2.80, and 2.90 Å, respectively. (11) This finding is consistent with stronger theoretical hydrogen bonding reported for P:Z (41) than G:C or A:T. P:Z pairs have also been shown to be more stable than possible mispairs experimentally. (42) Finally, the G:C pair in position 6 exhibits one long hydrogen bond for position 6, O6–N4 distance of 3.37 Å, potentially due to a combination of shearing by −1.4 Å, buckling of 5.4°, and opening of 12.6°. For position 7, all three hydrogen bonds are 2.71 Å. Thus, in the absence of a large shearing effect, G:C hydrogen bonding interactions are all less than 3.0 Å. The two P:Z pairs are also sheared by −0.87 and 0.84 Å, respectively, for positions 6 and 7, but retain normal hydrogen bonding distances.
另一方面，N2-O2、N1-N3 和 O6-N4 在 6 位的 P：Z 氢键距离分别为 2.57、2.79 和 2.94 Å，7 位 P：Z 的值分别为 2.63、2.80 和 2.90 Å。（11） 这一发现与报道的 P：Z （41） 比 G：C 或 A：T 更强的理论氢键一致。（42） 最后，位置 6 的 G：C 对在位置 6 处表现出一个长氢键，O6–N4 距离为 3.37 Å，这可能是由于 −1.4 Å 的剪切、5.4° 的屈曲和 12.6° 的开口的组合。对于位置 7，所有三个氢键均为 2.71 Å。因此，在没有大剪切效应的情况下，G：C 氢键相互作用都小于 3.0 Å。两个 P：Z 对在位置 6 和 7 也分别被 −0.87 和 0.84 Å 剪切，但保持正常的氢键距离。
另一方面，N2-O2、N1-N3 和 O6-N4 在 6 位的 P：Z 氢键距离分别为 2.57、2.79 和 2.94 Å，7 位 P：Z 的值分别为 2.63、2.80 和 2.90 Å。（11） 这一发现与报道的 P：Z （41） 比 G：C 或 A：T 更强的理论氢键一致。（42） 最后，位置 6 的 G：C 对在位置 6 处表现出一个长氢键，O6–N4 距离为 3.37 Å，这可能是由于 −1.4 Å 的剪切、5.4° 的屈曲和 12.6° 的开口的组合。对于位置 7，所有三个氢键均为 2.71 Å。因此，在没有大剪切效应的情况下，G：C 氢键相互作用都小于 3.0 Å。两个 P：Z 对在位置 6 和 7 也分别被 −0.87 和 0.84 Å 剪切，但保持正常的氢键距离。
A comparison with other local base pair step parameters of the 2X2K structure with other B-form DNA structures (G:C, A:T and P:Z), including axial rise per nucleotide (B-DNA values range between 3.03 Å to 3.37 Å) and base pair tilt (B-DNA values range between −5.9° to −16.4°), (31-34) shows that both rise and tilt values for 2X2K lie in the same range. The XX/KK dinucleotide step base has a rise and tilt of 3.43 Å and −5.12°. The phosphate–phosphate (P–P) distances also show little deviation from that of the standard B-form DNA. The P–P distance for B-DNA is around 7 Å, while for XX and KK, the P–P distances are 6.5 and 6.3 Å, respectively. (34) The chi angles of all the residues fall in the anticonformation as found in B-form DNA. Our results suggest that the 2X2K structure resembles B-form DNA, specifically B-form DNA sequences rich in G:C base pairs.
与 2X2K 结构与其他 B 型 DNA 结构（G：C、A：T 和 P：Z）的其他局部碱基对步骤参数的比较，包括每个核苷酸的轴向上升（B-DNA 值范围在 3.03 Å 至 3.37 Å 之间）和碱基对倾斜（B-DNA 值范围在 -5.9° 至 -16.4°之间），（31-34） 表明 2X2K 的上升和倾斜值位于同一范围内。XX/KK 二核苷酸阶梯基座的上升和倾斜为 3.43 Å 和 −5.12°。磷酸盐-磷酸盐 （P-P） 距离也与标准 B 型 DNA 的偏差很小。B-DNA 的 P-P 距离约为 7 Å，而 XX 和 KK 的 P-P 距离分别为 6.5 和 6.3 Å。（34） 所有残基的气角都属于 B 型 DNA 中的反构象。我们的结果表明，2X2K 结构类似于 B 型 DNA，特别是富含 G：C 碱基对的 B 型 DNA 序列。
与 2X2K 结构与其他 B 型 DNA 结构（G：C、A：T 和 P：Z）的其他局部碱基对步骤参数的比较，包括每个核苷酸的轴向上升（B-DNA 值范围在 3.03 Å 至 3.37 Å 之间）和碱基对倾斜（B-DNA 值范围在 -5.9° 至 -16.4°之间），（31-34） 表明 2X2K 的上升和倾斜值位于同一范围内。XX/KK 二核苷酸阶梯基座的上升和倾斜为 3.43 Å 和 −5.12°。磷酸盐-磷酸盐 （P-P） 距离也与标准 B 型 DNA 的偏差很小。B-DNA 的 P-P 距离约为 7 Å，而 XX 和 KK 的 P-P 距离分别为 6.5 和 6.3 Å。（34） 所有残基的气角都属于 B 型 DNA 中的反构象。我们的结果表明，2X2K 结构类似于 B 型 DNA，特别是富含 G：C 碱基对的 B 型 DNA 序列。
The Bigger Picture. Why Terran DNA Uses the Standard Nucleobase Pairs
更大的图景。为什么 Terran DNA 使用标准核碱基对
As experimental work associated with AEGIS pairs has developed, we have learned that pairs with nonstandard hydrogen bonding arrangements are able to robustly support duplex structures. This includes duplexes between strands where multiple consecutive nonstandard pairs are present. This is not the case for pairs whose pairing principle is based solely on hydrophobic interactions or size complementarity, lacking interbase hydrogen bonding. One example is Romesberg’s nonstandard bases d5SICS and dNaM that pair by hydrophobic and geometric complementarity and stack via an intercalative mode rather than edge on in duplex DNA. (16, 43) Another is Hirao’s hydrophobic unnatural base pair system between 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) and 2-nitro-4-propynylpyrrole (Px). (44, 45) Hirao’s hydrophobic nucleobases Ds:Px are successfully amplified in a PCR reaction; and the crystal structure of the ternary complex of Klentaq incorporating dPxTP opposite Ds indicates that the hydrophobic pairs can also act as promising candidates for incorporation by DNA polymerases. (46) However, these studies do not describe the effect of multiple hydrophobic base pairs on the overall structure of DNA duplex. A clash of templating dDs with side chain oxygen atom of Thr664, more flexible thumb domain in the ternary structure and lack of a binary structure incorporating these hydrophobic nucleotides suggest that more studies would be needed for hydrophobic base pairs before their use for the expansion of the genetic alphabet. (46)
随着与 AEGIS 对相关的实验工作的发展，我们了解到具有非标准氢键排列的对能够稳健地支持双相结构。这包括存在多个连续非标准对的链之间的双工。对于配对原理仅基于疏水相互作用或尺寸互补性、缺乏碱基间氢键的对，情况并非如此。一个例子是 Romesberg 的非标准碱基 d5SICS 和 dNaM，它们通过疏水和几何互补性配对，并通过嵌入模式堆叠，而不是在双链 DNA 中边缘堆叠。（16， 43）另一个是 7-（2-噻吩基）咪唑[4,5-b]吡啶 （Ds） 和 2-硝基-4-丙炔基吡咯 （Px） 之间的 Hirao 疏水性非自然碱基对系统。（44， 45）Hirao 的疏水性核碱基 Ds：Px 在 PCR 反应中成功扩增;Klentaq 的三元复合物的晶体结构掺入 dPxTP 与 Ds 相反，表明疏水对也可以作为 DNA 聚合酶掺入的有希望的候选者。（46） 然而，这些研究并未描述多个疏水碱基对对 DNA 双链体整体结构的影响。模板 dD 与 Thr664 的侧链氧原子的冲突、三元结构中更灵活的拇指结构域以及缺乏包含这些疏水核苷酸的二元结构表明，在将疏水碱基对用于扩展遗传字母表之前，需要对疏水碱基对进行更多研究。（46）
随着与 AEGIS 对相关的实验工作的发展，我们了解到具有非标准氢键排列的对能够稳健地支持双相结构。这包括存在多个连续非标准对的链之间的双工。对于配对原理仅基于疏水相互作用或尺寸互补性、缺乏碱基间氢键的对，情况并非如此。一个例子是 Romesberg 的非标准碱基 d5SICS 和 dNaM，它们通过疏水和几何互补性配对，并通过嵌入模式堆叠，而不是在双链 DNA 中边缘堆叠。（16， 43）另一个是 7-（2-噻吩基）咪唑[4,5-b]吡啶 （Ds） 和 2-硝基-4-丙炔基吡咯 （Px） 之间的 Hirao 疏水性非自然碱基对系统。（44， 45）Hirao 的疏水性核碱基 Ds：Px 在 PCR 反应中成功扩增;Klentaq 的三元复合物的晶体结构掺入 dPxTP 与 Ds 相反，表明疏水对也可以作为 DNA 聚合酶掺入的有希望的候选者。（46） 然而，这些研究并未描述多个疏水碱基对对 DNA 双链体整体结构的影响。模板 dD 与 Thr664 的侧链氧原子的冲突、三元结构中更灵活的拇指结构域以及缺乏包含这些疏水核苷酸的二元结构表明，在将疏水碱基对用于扩展遗传字母表之前，需要对疏水碱基对进行更多研究。（46）
To date, the vast majority of functional and structural data existing for nonstandard nucleobases mainly focuses on the incorporation of a single non-natural base pair in a DNA duplex. It is important to understand the effect of incorporation of multiple nonstandard base pairs in a duplex DNA that will not distort the overall structure of DNA significantly and also provide a basis for their retention in duplex DNA after multiple rounds of replication. Thus, the X:K pair meets a goal of synthetic biology, to develop expanded genetic systems that retain the desirable properties of natural nucleobases, including full evolvability.
迄今为止，存在的绝大多数非标准核碱基的功能和结构数据主要集中在 DNA 双链体中单个非天然碱基对的掺入。了解在双链 DNA 中掺入多个非标准碱基对的效果非常重要，它不会显著扭曲 DNA 的整体结构，并且还为它们在多轮复制后保留在双链 DNA 中提供了基础。因此，X：K 对满足了合成生物学的目标，即开发扩展的遗传系统，保留天然核碱基的理想特性，包括完全进化性。
迄今为止，存在的绝大多数非标准核碱基的功能和结构数据主要集中在 DNA 双链体中单个非天然碱基对的掺入。了解在双链 DNA 中掺入多个非标准碱基对的效果非常重要，它不会显著扭曲 DNA 的整体结构，并且还为它们在多轮复制后保留在双链 DNA 中提供了基础。因此，X：K 对满足了合成生物学的目标，即开发扩展的遗传系统，保留天然核碱基的理想特性，包括完全进化性。
These observations, however, also raise the question as to why natural DNA uses the nucleobases that it does. For example, we struggle to understand why adenine presents only two hydrogen bonding units to its thymine complement, while advanced biotechnologists must struggle to obtain uniform hybridization and priming in a system that contains a “weak” nucleobase pair and a “strong” nucleobase pair. Why not use 2-aminoadenine (diaminopurine) instead of adenine and get a pair joined by three canonical hydrogen bonds?
然而，这些观察结果也提出了一个问题，即为什么天然 DNA 使用它的核碱基。例如，我们很难理解为什么腺嘌呤只向其胸腺嘧啶互补体提供两个氢键单元，而高级生物技术专家必须努力在包含“弱”核碱基对和“强”核碱基对的系统中获得均匀的杂交和引发。为什么不使用 2-氨基腺嘌呤（二氨基嘌呤）代替腺嘌呤，并获得一对由三个经典氢键连接的氢键呢？
然而，这些观察结果也提出了一个问题，即为什么天然 DNA 使用它的核碱基。例如，我们很难理解为什么腺嘌呤只向其胸腺嘧啶互补体提供两个氢键单元，而高级生物技术专家必须努力在包含“弱”核碱基对和“强”核碱基对的系统中获得均匀的杂交和引发。为什么不使用 2-氨基腺嘌呤（二氨基嘌呤）代替腺嘌呤，并获得一对由三个经典氢键连接的氢键呢？
Under a Darwinian model, one cannot speak of “optimization” of a genetic system without presuming that alternative systems were accessible through random variation. In fact, some viruses are known to use 2-aminoadenine in their DNA, suggesting that this alternative was in fact available to terran life during its natural history. (47) Given this, one might interpret the surprisingly weak pair between 2-aminoadenine and thymidine, especially in DNA as compared to RNA, (48) as a second example of the disadvantage of symmetry in Watson–Crick pairing. This disadvantage is also present in the similarly symmetrical X:K pair.
在达尔文模型下，如果不假设替代系统可以通过随机变异获得，就不能谈论遗传系统的“优化”。事实上，已知一些病毒在其 DNA 中使用 2-氨基腺嘌呤，这表明这种替代品实际上在其自然历史中可供地球生命使用。（47） 鉴于此，人们可能会将 2-氨基腺嘌呤和胸苷之间出奇的弱对解释，尤其是在 DNA 中与 RNA 相比，（48） 作为 Watson-Crick 配对中对称性缺点的第二个例子。这个缺点也存在于类似对称的 X：K 对中。
在达尔文模型下，如果不假设替代系统可以通过随机变异获得，就不能谈论遗传系统的“优化”。事实上，已知一些病毒在其 DNA 中使用 2-氨基腺嘌呤，这表明这种替代品实际上在其自然历史中可供地球生命使用。（47） 鉴于此，人们可能会将 2-氨基腺嘌呤和胸苷之间出奇的弱对解释，尤其是在 DNA 中与 RNA 相比，（48） 作为 Watson-Crick 配对中对称性缺点的第二个例子。这个缺点也存在于类似对称的 X：K 对中。
Conclusion 结论
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In summary, properties of interest associated with the X:K pair include a unique pattern of hydrogen-bond donor and acceptors presented in the major and minor grooves that differs from those of A:T, G:C, or P:Z. As we observed for P:Z, inclusion of two consecutive X:K base pairs and four X:K pairs total in a self-complementary 16-mer oligonucleotide is readily accommodated in B-form DNA within our host–guest system. It is essential that any artificial components be accommodated in B-form DNA, as that is the form in which genomic DNA is most often found in cells. Sequence-specific properties associated with dinucleotide steps independent of their position in the oligonucleotide (excluding the three terminal base pairs involved in interactions with the protein) were previously demonstrated for a host–guest study of the CA dinucleotide integrase processing site. (24) Thus, the host–guest system has been vetted for analysis of sequence specific effects free of differing lattice constraints that might impact the structural properties of the oligonucleotides.
总之，与 X：K 对相关的感兴趣特性包括出现在大沟和小沟中的独特氢键供体和受体模式，这与 A：T、G：C 或 P：Z 不同。正如我们观察到的 P：Z，包括两个连续的 X：K 碱基对和四个 X：自互补的 16 聚体寡核苷酸中的总 K 对很容易适应我们的宿主-客体系统的 B 型 DNA 中。B 型 DNA 中必须包含任何人工成分，因为这是基因组 DNA 在细胞中最常见的形式。与二核苷酸步骤相关的序列特异性特性与它们在寡核苷酸中的位置无关（不包括与蛋白质相互作用所涉及的三个末端碱基对）先前已在 CA 二核苷酸整合酶加工位点的宿主-客体研究中得到证明。（24） 因此，已经审查了主客体系统，用于分析序列特异性效应，而没有可能影响寡核苷酸结构性质的不同晶格约束。
总之，与 X：K 对相关的感兴趣特性包括出现在大沟和小沟中的独特氢键供体和受体模式，这与 A：T、G：C 或 P：Z 不同。正如我们观察到的 P：Z，包括两个连续的 X：K 碱基对和四个 X：自互补的 16 聚体寡核苷酸中的总 K 对很容易适应我们的宿主-客体系统的 B 型 DNA 中。B 型 DNA 中必须包含任何人工成分，因为这是基因组 DNA 在细胞中最常见的形式。与二核苷酸步骤相关的序列特异性特性与它们在寡核苷酸中的位置无关（不包括与蛋白质相互作用所涉及的三个末端碱基对）先前已在 CA 二核苷酸整合酶加工位点的宿主-客体研究中得到证明。（24） 因此，已经审查了主客体系统，用于分析序列特异性效应，而没有可能影响寡核苷酸结构性质的不同晶格约束。
In this study, we analyzed the structural properties of the dinucleotide base pair steps for positions 6 and 7 including X:K, P:Z, G:C, or A:T. Structurally, the X:K pairs exhibit propeller angles similar to those in A:T pairs, while buckle values and other base pair parameter values were similar to those of G:C in the same position. The propensity for X:K pairs to exhibit significant propeller twist angles is supported by melting temperature data and calculation of hydrogen bonding energies for X:K, which are in fact weaker than A:T. These calculations support a hydrogen bonding pair of X:K comprising a deprotonated X and protonated K. Overall, the helical properties of 2X2K are B-form and most similar to G:C and P:Z with major and minor groove widths similar to those observed for G:C.
在这项研究中，我们分析了位置 6 和 7 的二核苷酸碱基对步骤的结构特性，包括 X：K、P：Z、G：C 或 A：T。在结构上，X：K 对表现出与 A：T 对相似的螺旋桨角度，而扣值和其他碱基对参数值与 G 相似：C 处于同一位置。X：K 对表现出显着螺旋桨扭曲角的倾向得到了 X：K 的熔化温度数据和氢键能计算的支持，X：K 实际上比 A：T 弱。这些计算支持由去质子化 X 和质子化 K 组成的氢键对 X：K。总体而言，2X2K 的螺旋特性为 B 型，与 G：C 和 P：Z 最相似，大槽和小槽宽度与 G：C 相似。
在这项研究中，我们分析了位置 6 和 7 的二核苷酸碱基对步骤的结构特性，包括 X：K、P：Z、G：C 或 A：T。在结构上，X：K 对表现出与 A：T 对相似的螺旋桨角度，而扣值和其他碱基对参数值与 G 相似：C 处于同一位置。X：K 对表现出显着螺旋桨扭曲角的倾向得到了 X：K 的熔化温度数据和氢键能计算的支持，X：K 实际上比 A：T 弱。这些计算支持由去质子化 X 和质子化 K 组成的氢键对 X：K。总体而言，2X2K 的螺旋特性为 B 型，与 G：C 和 P：Z 最相似，大槽和小槽宽度与 G：C 相似。
Thus, the inability of E. coli DNA polymerase I to incorporate two consecutive X:K pairs does not result from significant distortions in duplex DNA due to consecutive X:K pairs. Rather, it more likely results from the chemical properties of the nucleobases, the lack of a hydrogen bond acceptor in K, for example; DNA polymerases are known to “read” the minor groove through specific hydrogen bonding interactions. (25, 26) This is not a significant limitation for the use of X:K in expanding the genome as its use as a single pair is supported. In summary, we conclude that inclusion of X:K pairs provides unique properties while still maintaining compatibility with biologically relevant forms of DNA and therefore has the potential to expand the genetic alphabet.
因此，大肠杆菌 DNA 聚合酶 I 无法掺入两个连续的 X：K 对并不是由于连续的 X：K 对导致双链体 DNA 的显著扭曲造成的。相反，它更可能是由核碱基的化学性质引起的，例如 K 中缺乏氢键受体;已知 DNA 聚合酶通过特定的氢键相互作用“读取”小沟。（25， 26）这对于使用 X：K 扩展基因组来说并不是一个显着的限制，因为它支持将其用作单个对。总之，我们得出结论，包含 X：K 对提供了独特的特性，同时仍然保持与生物学相关形式的 DNA 的兼容性，因此有可能扩展遗传字母表。
因此，大肠杆菌 DNA 聚合酶 I 无法掺入两个连续的 X：K 对并不是由于连续的 X：K 对导致双链体 DNA 的显著扭曲造成的。相反，它更可能是由核碱基的化学性质引起的，例如 K 中缺乏氢键受体;已知 DNA 聚合酶通过特定的氢键相互作用“读取”小沟。（25， 26）这对于使用 X：K 扩展基因组来说并不是一个显着的限制，因为它支持将其用作单个对。总之，我们得出结论，包含 X：K 对提供了独特的特性，同时仍然保持与生物学相关形式的 DNA 的兼容性，因此有可能扩展遗传字母表。
Methods 方法
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Synthesis and Purification of KX Containing Oligonucleotides
含 KX 的寡核苷酸的合成和纯化
AEGIS-containing oligonucleotides were prepared by solid phase synthesis using phosphoramidites of dX and dK synthesized using procedures described elsewhere. These phosphoramidites are now available via Firebird Biomolecular Sciences, LLC (www.firebirdbio.com, Alachua, FL). The GACTKX “six letter” DNA molecules 5′-CTTATXXTAKKATAAG-3′ (2X2K) and 5′-CTTATXXXKKATAAG-3′ (3K3X) were prepared in house, as described below. The remaining sequences used here were purchased from IDT (Coralville IA).
使用使用其他地方描述的程序合成的 dX 和 dK 亚磷酰胺，通过固相合成制备含 AEGIS 的寡核苷酸。这些亚磷酰胺现在可通过 Firebird Biomolecular Sciences， LLC（www.firebirdbio.com，佛罗里达州阿拉楚阿）获得。GACTKX“六个字母”DNA 分子 5'-CTTATXXTAKKATAAG-3' （2X2K） 和 5'-CTTATXXXKKATAAG-3' （3K3X） 是在内部制备的，如下所述。此处使用的其余序列购自 IDT（爱荷华州克拉尔维尔）。
使用使用其他地方描述的程序合成的 dX 和 dK 亚磷酰胺，通过固相合成制备含 AEGIS 的寡核苷酸。这些亚磷酰胺现在可通过 Firebird Biomolecular Sciences， LLC（www.firebirdbio.com，佛罗里达州阿拉楚阿）获得。GACTKX“六个字母”DNA 分子 5'-CTTATXXTAKKATAAG-3' （2X2K） 和 5'-CTTATXXXKKATAAG-3' （3K3X） 是在内部制备的，如下所述。此处使用的其余序列购自 IDT（爱荷华州克拉尔维尔）。
Experiments showed that deprotection of GACTKX DNA oligonucleotides by treatment under standard conditions (ammonium hydroxide, 55 °C, overnight) led to substantial decomposition of the dX heterocycle. Therefore, the A, G, C, and K exocyclic amines were protected as the phenoxyacetyl, phenoxyacetyl, acetyl, and isobutyroyl groups, respectively. Then, all GACTKX oligonucleotides used in this study were deprotected using 50 mM K2CO3 in MeOH at 55 °C, overnight. They were then purified by HPLC.
实验表明，在标准条件（氢氧化铵，55 °C，过夜）下处理 GACTKX DNA 寡核苷酸后，其脱保护导致 dX 杂环大量分解。因此，A、G、C 和 K 外环胺分别以苯氧乙酰基、苯氧基乙酰基、乙酰基和异丁酰基的形式受到保护。然后，在 55 °C 下使用 50 mM K2CO3 的 MeOH 溶液去除本研究中使用的所有 GACTKX 寡核苷酸过夜。然后通过 HPLC 纯化它们。
实验表明，在标准条件（氢氧化铵，55 °C，过夜）下处理 GACTKX DNA 寡核苷酸后，其脱保护导致 dX 杂环大量分解。因此，A、G、C 和 K 外环胺分别以苯氧乙酰基、苯氧基乙酰基、乙酰基和异丁酰基的形式受到保护。然后，在 55 °C 下使用 50 mM K2CO3 的 MeOH 溶液去除本研究中使用的所有 GACTKX 寡核苷酸过夜。然后通过 HPLC 纯化它们。
Circular Dichroism Analyses
圆二色性分析
Circular dichroism (CD) studies were used to assess the helical form of the oligonucleotide duplexes in aqueous solution that was buffered at neutral pH with low salt concentrations. The self-pairing DNA sequences analyzed included 2X2K (5′-CTTATXXTAKKATAAG-3′), 3X3K (5′- CTTATXXXKKKATAAG-3′), the corresponding sequence with A:T pairs (5′-CTTATAAATTTATAAG-3′) and the corresponding sequence with G:C pairs (5′-CTTATGGGCCCATAAG-3′).
圆二色性 （CD） 研究用于评估寡核苷酸双链体在中性 pH 值和低盐浓度缓冲的水溶液中的螺旋形式。分析的自配对 DNA 序列包括 2X2K （5'-CTTATXXTAKKATAAG-3'）、3X3K （5'-CTTATXXXKKKATAAG-3'）、具有 A：T 对的相应序列 （5'-CTTATAAATTTATAAG-3'） 和具有 G：C 对的相应序列 （5'-CTTATGGGCCCATAAG-3'）。
圆二色性 （CD） 研究用于评估寡核苷酸双链体在中性 pH 值和低盐浓度缓冲的水溶液中的螺旋形式。分析的自配对 DNA 序列包括 2X2K （5'-CTTATXXTAKKATAAG-3'）、3X3K （5'-CTTATXXXKKKATAAG-3'）、具有 A：T 对的相应序列 （5'-CTTATAAATTTATAAG-3'） 和具有 G：C 对的相应序列 （5'-CTTATGGGCCCATAAG-3'）。
For CD analysis, stock solutions (2.5 mM) of these DNA sequences were diluted to 5 μM with buffer containing 10 mM HEPES pH 7.0 and 10 mM MgCl2. The CD spectra for DNA sequences were collected on a Jasco J-810 CD instrument at a temperature of 25 °C, at a rate of 50 nm/min and a wavelength increment of 0.1 nm. Ellipticity, Ø (mdegrees) was recorded for the DNA sequences from a wavelength of 320–220 nm. The final spectrum is the average of five scans corrected for ellipticity readings obtained for buffer (10 mM HEPES pH 7.0, 10 mM MgCl2) by itself. Spectra were initially measured for GC and AT control sequences and subsequently for 2X2K and 3X3K sequences.
对于 CD 分析，用含有 10 mM HEPES pH 7.0 和 10 mM MgCl2 的缓冲液将这些 DNA 序列的储备液 （2.5 mM） 稀释至 5 μM。DNA 序列的 CD 光谱在 Jasco J-810 CD 仪器上以 25 °C 的温度、50 nm/min 的速率和 0.1 nm 的波长增量收集。记录 320-220 nm 波长的 DNA 序列的椭圆度 Ø （mdegrees）。最终谱图是针对缓冲液（10 mM HEPES pH 7.0,10 mM MgCl2）自身获得的椭圆度读数进行校正的五次扫描的平均值。最初测量 GC 和 AT 对照序列的谱图，随后测量 2X2K 和 3X3K 序列的谱图。
对于 CD 分析，用含有 10 mM HEPES pH 7.0 和 10 mM MgCl2 的缓冲液将这些 DNA 序列的储备液 （2.5 mM） 稀释至 5 μM。DNA 序列的 CD 光谱在 Jasco J-810 CD 仪器上以 25 °C 的温度、50 nm/min 的速率和 0.1 nm 的波长增量收集。记录 320-220 nm 波长的 DNA 序列的椭圆度 Ø （mdegrees）。最终谱图是针对缓冲液（10 mM HEPES pH 7.0,10 mM MgCl2）自身获得的椭圆度读数进行校正的五次扫描的平均值。最初测量 GC 和 AT 对照序列的谱图，随后测量 2X2K 和 3X3K 序列的谱图。
Crystallization and Data Collection
结晶和数据收集
The self-complementary 16-mer DNA oligonucleotides containing either two or three X:K pairs (2X2K or 3X3K) were resuspended in buffer containing 10 mM HEPES (pH 7.0) and 10 mM MgCl2 and then annealed by heating to 70 °C followed by slow cooling to room temperature to give a final concentration of 2.5 mM duplex DNA. The protein (the N-terminal fragment including residues 24–278 of Moloney murine leukemia virus reverse transcriptase, MMLV RT) was diluted to a concentration of 0.65 mM in two steps. A 2.9 mM stock solution of the protein was diluted to 1.4 mM using 50 mM MES (pH 6.0) and 0.3 M NaCl. This 1.4 mM substock was then further diluted to 0.65 mM in 100 mM HEPES (pH 7.5) and 0.3 M NaCl. The host–2X2K or host–3X3K (protein–DNA) complex was set at a ratio of 1:2 respectively (0.43 mM protein/ 0.86 mM DNA) in buffer containing 100 mM HEPES (pH 7.5), 0.3 M NaCl and incubated at 4 °C for 1 h.
将含有 2 对或 3 对 X：K（2X2K 或 3X3K）的自互补 16 聚体 DNA 寡核苷酸重悬于含有 10 mM HEPES （pH 7.0） 和 10 mM MgCl2 的缓冲液中，然后加热至 70 °C 进行退火，然后缓慢冷却至室温，得到最终浓度为 2.5 mM 的双链体 DNA。分两步将蛋白质（包括 Moloney 鼠白血病病毒逆转录酶 MMLV RT 残基 24-278 的 N 末端片段）稀释至 0.65 mM 的浓度。使用 50 mM MES （pH 6.0） 和 0.3 M NaCl 将 2.9 mM 蛋白质储备液稀释至 1.4 mM。然后将该 1.4 mM 亚储备液在 100 mM HEPES （pH 7.5） 和 0.3 M NaCl 中进一步稀释至 0.65 mM。将宿主–2X2K 或宿主–3X3K（蛋白质–DNA）复合物分别以 1：2 的比例（0.43 mM 蛋白质/0.86 mM DNA）设置在含有 100 mM HEPES （pH 7.5）、0.3 M NaCl 的缓冲液中，并在 4 °C 下孵育 1 小时。
将含有 2 对或 3 对 X：K（2X2K 或 3X3K）的自互补 16 聚体 DNA 寡核苷酸重悬于含有 10 mM HEPES （pH 7.0） 和 10 mM MgCl2 的缓冲液中，然后加热至 70 °C 进行退火，然后缓慢冷却至室温，得到最终浓度为 2.5 mM 的双链体 DNA。分两步将蛋白质（包括 Moloney 鼠白血病病毒逆转录酶 MMLV RT 残基 24-278 的 N 末端片段）稀释至 0.65 mM 的浓度。使用 50 mM MES （pH 6.0） 和 0.3 M NaCl 将 2.9 mM 蛋白质储备液稀释至 1.4 mM。然后将该 1.4 mM 亚储备液在 100 mM HEPES （pH 7.5） 和 0.3 M NaCl 中进一步稀释至 0.65 mM。将宿主–2X2K 或宿主–3X3K（蛋白质–DNA）复合物分别以 1：2 的比例（0.43 mM 蛋白质/0.86 mM DNA）设置在含有 100 mM HEPES （pH 7.5）、0.3 M NaCl 的缓冲液中，并在 4 °C 下孵育 1 小时。
The self-nucleated protein–DNA crystals of host–GC were used as seeds for crystallizing host protein with oligonucleotides containing KX. Crystals of host protein complexed with GC grew in hanging drops containing 1 μL of protein–DNA complex and 1 μL of solution containing 10% PEG 4000, 5 mM magnesium acetate, and 50 mM ADA (pH 6.5). The reservoir solution consisted of 500 μL of 10% PEG 4000, 5 mM magnesium acetate, and 50 mM ADA (pH 6.5). Host–2X2K microseeded crystals grew at 8% PEG 4000, 5 mM magnesium acetate, and 50 mM ADA (pH 6.5). No host–guest crystals were obtained for the 3X3K complex. DNA-only 3X3K crystals did not diffract to high resolution and were not further pursued. The host–2X2K crystals were cryoprotected in 9% PEG 4000, 5 mM magnesium acetate, 100 mM HEPES (pH 7.5), and 20% ethylene glycol before flash freezing in liquid nitrogen for data collection.
使用宿主-GC 的自成核蛋白质-DNA 晶体作为种子，用含有 KX 的寡核苷酸结晶宿主蛋白质。与 GC 复合物结合的宿主蛋白晶体在含有 1 μL 蛋白质-DNA 复合物和 1 μL 含有 10% PEG 4000、5 mM 乙酸镁和 50 mM ADA （pH 6.5） 的溶液的悬滴中生长。储液槽溶液由 500 μL 10% PEG 4000、5 mM 乙酸镁和 50 mM ADA （pH 6.5） 组成。宿主–2X2K 微晶种晶体在 8% PEG 4000、5 mM 乙酸镁和 50 mM ADA （pH 6.5） 下生长。没有获得 3X3K 复合物的主客体晶体。仅 DNA 的 3X3K 晶体没有衍射到高分辨率，因此没有被进一步追求。在 9% PEG 4000、5 mM 乙酸镁、100 mM HEPES （pH 7.5） 和 20% 乙二醇中冷冻保护宿主–2X2K 晶体，然后在液氮中快速冷冻以进行数据收集。
使用宿主-GC 的自成核蛋白质-DNA 晶体作为种子，用含有 KX 的寡核苷酸结晶宿主蛋白质。与 GC 复合物结合的宿主蛋白晶体在含有 1 μL 蛋白质-DNA 复合物和 1 μL 含有 10% PEG 4000、5 mM 乙酸镁和 50 mM ADA （pH 6.5） 的溶液的悬滴中生长。储液槽溶液由 500 μL 10% PEG 4000、5 mM 乙酸镁和 50 mM ADA （pH 6.5） 组成。宿主–2X2K 微晶种晶体在 8% PEG 4000、5 mM 乙酸镁和 50 mM ADA （pH 6.5） 下生长。没有获得 3X3K 复合物的主客体晶体。仅 DNA 的 3X3K 晶体没有衍射到高分辨率，因此没有被进一步追求。在 9% PEG 4000、5 mM 乙酸镁、100 mM HEPES （pH 7.5） 和 20% 乙二醇中冷冻保护宿主–2X2K 晶体，然后在液氮中快速冷冻以进行数据收集。
The 2X2K host–guest complex data were collected to a resolution of 1.75 Å at SBC-19-BM beamline at the Advanced Photon Source, Argonne National Laboratory, Darien, IL. Data reduction was carried out in space group P21212, and data were indexed, integrated, and scaled using HKL3000. (49) The host–guest crystal structure was determined by molecular replacement using the CCP4 program MOLREP (50) using the protein model from PDB ID 4XO0. (11) Use of the protein model alone for phasing provided unbiased electron density for the DNA in the host guest complex. Adjustments to the protein model and addition of water molecules were done in COOT (51) and initially refined in REFMAC. (52)
在伊利诺伊州达里恩阿贡国家实验室高级光子源的 SBC-19-BM 光束线上以 1.75 Å 的分辨率收集 2X2K 主客体复合物数据。数据缩减在空间群 P2121 2中进行，并使用 HKL3000对数据进行索引、整合和缩放。（49） 使用 PDB ID 4XO0 的蛋白质模型，使用 CCP4 程序 MOLREP （50） 通过分子置换来确定主客体晶体结构。（11） 单独使用蛋白质模型进行定相为宿主客体复合物中的 DNA 提供了无偏电子密度。在 COOT （51） 中对蛋白质模型的调整和水分子的添加，最初在 REFMAC 中精制。（52）
在伊利诺伊州达里恩阿贡国家实验室高级光子源的 SBC-19-BM 光束线上以 1.75 Å 的分辨率收集 2X2K 主客体复合物数据。数据缩减在空间群 P2121 2中进行，并使用 HKL3000对数据进行索引、整合和缩放。（49） 使用 PDB ID 4XO0 的蛋白质模型，使用 CCP4 程序 MOLREP （50） 通过分子置换来确定主客体晶体结构。（11） 单独使用蛋白质模型进行定相为宿主客体复合物中的 DNA 提供了无偏电子密度。在 COOT （51） 中对蛋白质模型的调整和水分子的添加，最初在 REFMAC 中精制。（52）
For building the DNA, initially 3 base pairs were built, followed by refinement in REFMAC to improve the electron density for the next consecutive base pairs. The protein–DNA refinement was followed by the addition of two more base pairs and refinement in REFMAC. The parameter and linking files for KX were created in PHENIX. (53) Finally, the last three base pairs were added including the Ks and Xs, and refinement was done using PHENIX. Multiple rounds of model adjustment in COOT and refinement in PHENIX yielded R-work and R-free values of 20.86% and 23.85%, respectively. Coordinates have been deposited for 2X2K with PDB (Table 1) with PDB identifier 5VBS.
为了构建 DNA，最初构建 3 个碱基对，然后在 REFMAC 中精炼以提高下一个连续碱基对的电子密度。蛋白质-DNA 精制之后，再添加两个碱基对并在 REFMAC 中精制。KX 的参数和链接文件是在 PHENIX 中创建的。（53） 最后，添加了最后三个碱基对，包括 Ks 和 Xs，并使用 PHENIX 进行了细化。在 COOT 中进行多轮模型调整，在 PHENIX 中进行细化，R-work 和 R-free 值分别为 20.86% 和 23.85%。PDB 的坐标已存入 2X2K（表 1），PDB 标识符为 5VBS。
为了构建 DNA，最初构建 3 个碱基对，然后在 REFMAC 中精炼以提高下一个连续碱基对的电子密度。蛋白质-DNA 精制之后，再添加两个碱基对并在 REFMAC 中精制。KX 的参数和链接文件是在 PHENIX 中创建的。（53） 最后，添加了最后三个碱基对，包括 Ks 和 Xs，并使用 PHENIX 进行了细化。在 COOT 中进行多轮模型调整，在 PHENIX 中进行细化，R-work 和 R-free 值分别为 20.86% 和 23.85%。PDB 的坐标已存入 2X2K（表 1），PDB 标识符为 5VBS。
Quantum Mechanical Calculations
量子力学计算
Structures of the X:K, P:Z, G:C, and A:T pairs were optimized using M06-2X (54)/aug″-cc-pVTZ (55) in the Gaussian 09 software. (56) In each structure, the 2′-deoxyribose was modeled by a methyl group to represent the surrounding electronic environment. The single prime notation refers to the use of aug′-cc-pVTZ on all heavy atoms (not hydrogen); the use of double primes indicates that no diffuse functions were used on either hydrogen or carbon atoms. This approach was chosen due to the linear dependencies present by having so many diffuse functions over aromatic rings, indicating that their absence helps SCF convergence with no loss in the basis representation.
在 Gaussian 09 软件中使用 M06-2X （54）/aug“-cc-pVTZ （55） 优化了 X：K、P：Z、G：C 和 A：T 对的结构。（56） 在每个结构中，2'-脱氧核糖由甲基建模以表示周围的电子环境。单素数符号是指在所有重原子（不是氢）上使用 aug′-cc-pVTZ;使用双素数表明没有对氢原子或碳原子使用漫反射函数。选择这种方法是因为芳香环上具有如此多的扩散函数存在线性依赖性，这表明它们的缺失有助于 SCF 收敛，而不会损失基表示。
在 Gaussian 09 软件中使用 M06-2X （54）/aug“-cc-pVTZ （55） 优化了 X：K、P：Z、G：C 和 A：T 对的结构。（56） 在每个结构中，2'-脱氧核糖由甲基建模以表示周围的电子环境。单素数符号是指在所有重原子（不是氢）上使用 aug′-cc-pVTZ;使用双素数表明没有对氢原子或碳原子使用漫反射函数。选择这种方法是因为芳香环上具有如此多的扩散函数存在线性依赖性，这表明它们的缺失有助于 SCF 收敛，而不会损失基表示。
A structure was deemed to be “optimized” when the RMS force was no greater than 1.0 × 10–4 Hartree/Bohr over all geometric parameters and no single geometric parameter had a force greater than 3.3 × 10–4 Hartree/Bohr. The KS-DFT (57, 58) Lebedev integration grid used 99 radial points and 590 solid angle points. The SCF was deemed to be “converged” when the change in SCF matrix elements was less than 10–6. Spherical d functions were used throughout for all basis sets in this paper. The construction of all electrostatic potential maps used the KS determinant density with isocontours 0.001 elementary charge per cubic Bohr. The M06-2X functional was chosen for its strong performance in calculating organic molecule energies, geometries, and dispersion phenomena, despite being parametrized empirically. All dipole moments calculated were based on this SCF reference.
当 RMS 力在所有几何参数上不大于 1.0 × 10-4 Hartree/Bohr 并且没有单个几何参数的力大于 3.3 × 10-4 Hartree/Bohr 时，结构被认为是“优化的”。KS-DFT （57， 58） Lebedev 积分网格使用了 99 个径向点和 590 个立体角点。当 SCF 矩阵元素的变化小于 10-6 时，SCF 被认为是“收敛的”。在本文中，所有基集都使用了球形 d 函数。所有静电势图的构建都使用 KS 行列式密度，等值线为每立方玻尔 0.001 基本电荷。选择 M06-2X 泛函是因为其在计算有机分子能量、几何形状和色散现象方面的强大性能，尽管它是凭经验参数化的。计算的所有偶极矩均基于此 SCF 参考。
当 RMS 力在所有几何参数上不大于 1.0 × 10-4 Hartree/Bohr 并且没有单个几何参数的力大于 3.3 × 10-4 Hartree/Bohr 时，结构被认为是“优化的”。KS-DFT （57， 58） Lebedev 积分网格使用了 99 个径向点和 590 个立体角点。当 SCF 矩阵元素的变化小于 10-6 时，SCF 被认为是“收敛的”。在本文中，所有基集都使用了球形 d 函数。所有静电势图的构建都使用 KS 行列式密度，等值线为每立方玻尔 0.001 基本电荷。选择 M06-2X 泛函是因为其在计算有机分子能量、几何形状和色散现象方面的强大性能，尽管它是凭经验参数化的。计算的所有偶极矩均基于此 SCF 参考。
The Watson–Crick hydrogen bonding free energy utilized the composite method of CCSD(T) (37-39)/aug′-cc-pVDZ describing the electronic energy, which has been shown to be accurate to ∼1 kcal/mol of the true gas-phase energy of single-reference wave functions. (59) The basis set extrapolation used the Helgaker (36) scheme for aug″-cc-pVTZ and aug″-cc-pVQZ extrapolation. The coupled cluster equations were considered “converged” when the coupled cluster energy equation tensor amplitudes changed less than 10–6. All coupled cluster and MP2 calculations utilized the ACES3 software (60) for its ability to parallelize over thousands of processors. All calculations were performed on the Big Red II supercomputer of Indiana University. The entropic contributions to the free energy used the vibrational partition function based on M06-2X/aug″-cc-pVTZ optimization. The Watson–Crick hydrogen bond energy is here defined to be the difference between the interacting pair (say PZ) vs the lone fragments, each individually optimized (P and Z, separately, in this case).
Watson-Crick 氢键自由能利用 CCSD（T） （37-39）/aug′-cc-pVDZ 的复合方法描述电子能，该方法已被证明精确到单参考波函数的真实气相能量的 ∼1 kcal/mol。（59） 基集外推使用 Helgaker （36） 方案进行 aug“-cc-pVTZ 和 aug”-cc-pVQZ 外推。当耦合簇能量方程张量幅度变化小于 10-6 时，耦合簇方程被认为是“收敛的”。所有耦合的集群和 MP2 计算都使用 ACES3 软件 （60），因为它能够并行处理数千个处理器。所有计算均在印第安纳大学的 Big Red II 超级计算机上进行。熵对自由能的贡献使用了基于 M06-2X/aug“-cc-pVTZ 优化的振动分配函数。Watson-Crick 氢键能在这里定义为相互作用对（比如 PZ）与孤片段之间的差值，每个片段都单独优化（在本例中分别为 P 和 Z）。
Watson-Crick 氢键自由能利用 CCSD（T） （37-39）/aug′-cc-pVDZ 的复合方法描述电子能，该方法已被证明精确到单参考波函数的真实气相能量的 ∼1 kcal/mol。（59） 基集外推使用 Helgaker （36） 方案进行 aug“-cc-pVTZ 和 aug”-cc-pVQZ 外推。当耦合簇能量方程张量幅度变化小于 10-6 时，耦合簇方程被认为是“收敛的”。所有耦合的集群和 MP2 计算都使用 ACES3 软件 （60），因为它能够并行处理数千个处理器。所有计算均在印第安纳大学的 Big Red II 超级计算机上进行。熵对自由能的贡献使用了基于 M06-2X/aug“-cc-pVTZ 优化的振动分配函数。Watson-Crick 氢键能在这里定义为相互作用对（比如 PZ）与孤片段之间的差值，每个片段都单独优化（在本例中分别为 P 和 Z）。
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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssynbio.7b00150.
支持信息可在 ACS Publications 网站上免费获取，网址为 DOI：10.1021/acssynbio.7b00150。
Figure S1. UV absorbance as a function of pH for the dX nucleoside gives pKa of 8.5 ± 0.1. Table S1. Melting point temperatures for oligonucleotides containing X:K pairs Coordinates of M06-2X/aug″-cc-pVDZ optimized structures, Å (PDF)
图 S1.dX 核苷的紫外吸光度与 pH 值的函数关系，得到 pKa 为 8.5 ± 0.1。表 S1.含有 X：K 对的寡核苷酸的熔点温度 M06-2X/aug“-cc-pVDZ 优化结构的坐标，Å （PDF）
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Author Information 作者信息
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- Millie M. Georgiadis - Department of Biochemistry & Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202, United States; Department of Chemistry and Chemical Biology, Indiana University, Purdue University Indianapolis, Indianapolis, Indiana 46202, United States;http://orcid.org/0000-0003-2976-4576;
Millie M. Georgiadis - 印第安纳大学医学院生物化学与分子生物学系，印第安纳波利斯，印第安纳州46202，美国;普渡大学印第安纳波利斯分校化学与化学生物学系，印第安纳波利斯，印第安纳州 46202，美国; http://orcid.org/0000-0003-2976-4576;
- Steven A. Benner - Foundation for Applied Molecular Evolution, and the Westheimer Institute of Science & Technology, 13709 Progress Boulevard, Box 7, Alachua, Florida 32615, United States; Firebird Biomolecular Sciences LLC, 13709 Progress Boulevard, Box 17, Alachua, Florida 32615, United States;http://orcid.org/0000-0002-3318-9917
Steven A. Benner - 应用分子进化基金会，以及Westheimer科学与技术研究所，地址为13709 Progress Boulevard， Box 7， Alachua， Florida 32615， United States;Firebird Biomolecular Sciences LLC，13709 Progress Boulevard， Box 17， Alachua， Florida 32615， 美国; http://orcid.org/0000-0002-3318-9917
Acknowledgment 确认
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Results shown in this report are derived from work performed at Argonne National Laboratory, Structural Biology Center at the Advanced Photon Source. Argonne is operated by UChicago Argonne, LLC, for the U.S. Department of Energy, Office of Biological and Environmental Research under Contract DE-AC02-06CH11357. Calculations were performed on the Indiana University Big Red 2 supercomputer. The authors thank the Lilly Endowment, Inc. for support of the Indiana University Pervasive Technology Institute and the Indiana METACyt Initiative. The material is based in part upon work supported by NASA under awards NNX14AK37G and NNX15AF46G. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Aeronautics and Space Administration. Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R01GM111386. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. This material is also based in part upon work supported by the National Science Foundation under Grant No. CHE 1507816.
本报告中显示的结果来自阿贡国家实验室结构生物学中心在先进光子源进行的工作。Argonne 由 UChicago Argonne， LLC 为美国能源部生物与环境研究办公室运营，合同编号为 DE-AC02-06CH11357。在印第安纳大学 Big Red 2 超级计算机上执行计算。作者感谢 Lilly Endowment， Inc. 对印第安纳大学普适技术研究所和印第安纳州 METACyt 倡议的支持。该材料部分基于 NASA 在 NNX14AK37G 和 NNX15AF46G 奖项下支持的工作。本材料中表达的任何意见、发现、结论或建议均为作者的观点，并不一定反映美国国家航空航天局的观点。本出版物中报告的研究得到了美国国立卫生研究院国家普通医学科学研究所的支持，奖项编号为 R01GM111386。内容完全由作者负责，并不一定代表 NIH 的官方观点。本材料还部分基于美国国家科学基金会 （National Science Foundation） 在 Grant No.CHE 1507816。
References 引用
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本文引用了其他 60 种出版物。
- 1Ghosh, A. and Bansal, M. (2003) A glossary of DNA structures from A to Z Acta Crystallogr., Sect. D: Biol. Crystallogr. 59, 620 DOI: 10.1107/S0907444903003251Google Scholar 谷歌学术1https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXit12kt7Y%253D&md5=9d1306e34c1930e36d870b596f28ee55A glossary of DNA structures from A to ZGhosh, Anirban; Bansal, ManjuActa Crystallographica, Section D: Biological Crystallography (2003), D59 (4), 620-626CODEN: ABCRE6; ISSN:0907-4449. (Blackwell Munksgaard)A review. The right-handed double-helical Watson-Crick model for B-form DNA is the most commonly known DNA structure. In addn. to this classic structure, several other forms of DNA have been obsd., and it is clear that the DNA mol. can assume different structures depending on the base sequence and environment. The various forms of DNA have been identified as A, B, C etc. In fact, a detailed inspection of the literature reveals that only the letters F, Q, U, V and Y are now available to describe any new DNA structure that may appear in the future. It is also apparent that it may be more relevant to talk about the A, B or C type dinucleotide steps, since several recent structures show mixts. of various different geometries and a careful anal. is essential before identifying it as a 'new structure'. This review provides a glossary of currently identified DNA structures and is quite timely as it outlines the present understanding of DNA structure exactly 50 yr after the original discovery of DNA structure by Watson and Crick.
1高希，A. 和 Bansal， M. （2003） A glossary of DNA structures from A to Z Acta Crystallogr.， Sect. D： Biol. Crystallogr. 59， 620 DOI： 10.1107/S0907444903003251Google Scholar内容1https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXit12kt7Y%253D&md5=9d1306e34c1930e36d870b596f28ee55 的更多从 A 到 Z 的 DNA 结构词汇表Ghosh， Anirban;Bansal，ManjuActa Crystallographica，D 部分：生物晶体学 （2003 年）、 D59 系列 （4）、 620-626 元科登： ABCRE6; 国际标准书号：0907-4449。 （布莱克威尔·蒙克斯加德）评论。 B 型 DNA 的右旋双螺旋 Watson-Crick 模型是最常见的 DNA 结构。 除了这种经典结构之外，还观察到了其他几种形式的 DNA，很明显，DNA 分子可以根据碱基序列和环境呈现不同的结构。 各种形式的 DNA 已被鉴定为 A、B、C 等。 事实上，对文献的详细检查表明，现在只有字母 F、Q、U、V 和 Y 可用于描述未来可能出现的任何新 DNA 结构。 同样明显的是，谈论 A、B 或 C 型二核苷酸步骤可能更相关，因为最近的几个结构显示了混合物。在将其识别为“新结构”之前，必须进行仔细的肛门检查。 这篇综述提供了目前已确定的 DNA 结构的词汇表，并且非常及时，因为它概述了目前对 DNA 结构的理解，恰好是在 Watson 和 Crick 最初发现 DNA 结构 50 年后。 - 2Switzer, C., Moroney, S. E., and Benner, S. A. (1989) Enzymatic incorporation of a new base pair into DNA and RNA J. Am. Chem. Soc. 111, 8322 DOI: 10.1021/ja00203a067Google Scholar 谷歌学术2https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1MXls12mtr8%253D&md5=53ace25f0d8cc4aa3a6be39a90a328c0Enzymatic incorporation of a new base pair into DNA and RNASwitzer, Christopher; Moroney, Simon E.; Benner, Steven A.Journal of the American Chemical Society (1989), 111 (21), 8322-3CODEN: JACSAT; ISSN:0002-7863.The Klenow fragment of DNA polymerase I (Escherichia coli) and phage T7 RNA polymerase were found to direct the incorporation of isoguanosine (iso-G) into an oligonucleotide opposite isocytidine (iso-C). Further, expts. were carried out with the Klenow enzyme to det. the specificity with which the new bases pair. On the basis of these expts., it was detd. that essentially no deoxyguanosine or deoxyadenosine was incorporated opposite d-iso-C, and that whereas d-iso-G showed undesired pairing with deoxycytidine. Due to the specificity obsd. in the enzymic incorporation of d-iso-G into DNA, it was concluded that these 2 mols. form a base-pair with a H-bonding pattern distinct from those occurring in the natural A-T(U) and G-C pairs.
2瑞士 C.， 莫罗尼， SE和 Benner， S. A. （1989） 将新的碱基对酶促掺入 DNA 和 RNA 中 J. Am. Chem. Soc. 111， 8322 DOI： 10.1021/JA00203A067Google Scholar的更多内容2https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1MXls12mtr8%253D&md5=53ace25f0d8cc4aa3a6be39a90a328c0酶促掺入 DNA 和 RNA 中Switzer， Christopher;莫罗尼，西蒙 E.;本纳，史蒂文 A.美国化学会杂志 （1989 年）、 111 元 （21）、 8322-3科登： JACSAT公司; 国际标准书号：0002-7863。发现 DNA 聚合酶 I （大肠杆菌） 和噬菌体 T7 RNA 聚合酶的 Klenow 片段指导异鸟苷 （iso-G） 掺入与异胞苷 （iso-C） 相反的寡核苷酸中。 此外，expts.与 Klenow 酶一起进行，以确定新碱基配对的特异性。 根据这些 expits.，它是 detd.基本上没有脱氧鸟苷或脱氧腺苷与 d-iso-C 相反掺入，而 d-iso-G 显示出与脱氧胞苷的不希望配对。 由于特异性 obsd.在将 d-iso-G 酶促掺入 DNA 中时，得出结论这 2 mols.形成一个碱基对，其 H 键合模式与天然 AT（U） 和 GC 对中出现的模式不同。 - 3Piccirilli, J. A., Krauch, T., Moroney, S. E., and Benner, S. A. (1990) Enzymatic incorporation of a new base pair into DNA and RNA extends the genetic alphabet Nature 343, 33 DOI: 10.1038/343033a0Google Scholar 谷歌学术3https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3cXitFamu7k%253D&md5=1abf50139303a7ffa31262cdeb736fe5Enzymic incorporation of a new base pair into DNA and RNA extends the genetic alphabetPiccirilli, Joseph A.; Krauch, Tilman; Moroney, Simon E.; Benner, Steven A.Nature (London, United Kingdom) (1990), 343 (6253), 33-7CODEN: NATUAS; ISSN:0028-0836.A new Watson-Crick base pair [κ-xanthine or 7-Me oxyformycin B, where κ = 3-β-D-ribofuranosyl-(2,6-diaminopyrimidine) with a H bonding pattern different from that in the A·T and G·C base pairs, is incorporated into duplex DNA and RNA by DNA and RNA polymerases and expands the genetic alphabet from 4 to 6 letters. This expansion could lead to RNAs with greater diversity in functional groups and greater catalytic potential.
3皮奇里利，J. A.， 克劳奇， T.， 莫罗尼， SE和 Benner， S. A. （1990） 将新的碱基对酶促掺入 DNA 和 RNA 扩展了遗传字母表 Nature 343， 33 DOI： 10.1038/343033A0Google Scholar内容3 将新的碱基对酶促掺入 DNA 和 RNA 中，扩展了遗传字母表Piccirilli， Joseph A.;克劳赫，蒂尔曼;莫罗尼，西蒙 E.;本纳，史蒂文 A.Nature （英国 伦敦） （1990 年）、 343 元 （6253）、 33-7科登： 纳图阿斯; 国际标准书号：0028-0836。一个新的 Watson-Crick 碱基对 [κ-黄嘌呤或 7-Me 氧甲酰蛋白 B，其中 κ = 3-β-D-呋喃核糖基-（2,6-二氨基嘧啶），其 H 键合模式与 A·T 和 G·C 碱基对，通过 DNA 和 RNA 聚合酶掺入双链 DNA 和 RNA 中，并将遗传字母表从 4 个字母扩展到 6 个字母。 这种扩增可能导致 RNA 具有更大的功能基团多样性和更大的催化潜力。 - 4Benner, S. A. (2004) Understanding nucleic acids using synthetic chemistry Acc. Chem. Res. 37, 784 DOI: 10.1021/ar040004zGoogle Scholar 谷歌学术4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXntVOrsb8%253D&md5=ce508328dbf0543bdd7f3b79bda79b84Understanding Nucleic Acids Using Synthetic ChemistryBenner, Steven A.Accounts of Chemical Research (2004), 37 (10), 784-797CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)A review describing work done in these labs. that has used synthetic, phys. org., and biol. chem. to understand the roles played by the nucleobases, sugars, and phosphates of DNA in the mol. recognition processes central to genetics. The no. of nucleobases has been increased from 4 to 12, generating an artificially expanded genetic information system. This system is used today in the clinic to monitor the levels of HIV and hepatitis C viruses in patients, helping to manage patient care. Work with uncharged phosphate replacements suggests that a repeating charge is a universal feature of genetic mols. operating in water and will be found in extraterrestrial life (if it is ever encountered). The use of ribose may reflect prebiotic processes in the presence of borate-contg. minerals, which stabilize ribose formed from simple org. precursors. A new field, synthetic biol., is emerging on the basis of these expts., where chem. mimics biol. processes as complicated as Darwinian evolution.
4本纳，SA （2004） 使用合成化学了解核酸 Acc. Chem. Res. 37， 784 DOI： 10.1021/AR040004ZGoogle Scholar内容4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXntVOrsb8%253D&md5=ce508328dbf0543bdd7f3b79bda79b84 的更多使用合成化学了解核酸Benner， Steven A.化学研究账户 （2004 年）， 37 元 （10）、 784-797科登： 阿赫雷4; 国际标准书号：0001-4842。 （美国化学学会）一篇描述这些实验室所做工作的综述，使用 Synthetic、Phys. Org. 和 Biol. Chem. 来了解 DNA 的核碱基、糖和磷酸盐在遗传学核心的分子识别过程中所起的作用。 没有。的核碱基已从 4 个增加到 12 个，从而产生了一个人工扩展的遗传信息系统。 该系统如今在临床上用于监测患者的 HIV 和丙型肝炎病毒水平，帮助管理患者护理。 未带电的磷酸盐替代品的研究表明，重复电荷是遗传 mols 的普遍特征。在水中操作，会在外星生命中发现（如果遇到）。 核糖的使用可能反映了硼酸盐浓度存在下的益生元过程。矿物质，稳定由简单的组织前体形成的核糖。 在这些研究的基础上，一个新的领域，合成生物学正在出现，其中化学模仿了像达尔文进化论一样复杂的生物学过程。 - 5Geyer, C. R., Battersby, T. R., and Benner, S. A. (2003) Nucleobase pairing in expanded Watson-Crick-like genetic information systems Structure 11, 1485 DOI: 10.1016/j.str.2003.11.008Google Scholar 谷歌学术5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXps1OrtL0%253D&md5=4fdcf5a3d34c73b118c90f57246cbc11Nucleobase Pairing in Expanded Watson-Crick-like Genetic Information SystemsGeyer, C. Ronald; Battersby, Thomas R.; Benner, Steven A.Structure (Cambridge, MA, United States) (2003), 11 (12), 1485-1498CODEN: STRUE6; ISSN:0969-2126. (Cell Press)To guide the design of alternative genetic systems, we measured melting temps. of DNA duplexes contg. matched and mismatched nucleobase pairs from natural and unnatural structures. The pairs were analyzed in terms of structural features, including nucleobase size, no. of hydrogen bonds formed, the presence of uncompensated hydrogen bonding functional groups, the nature of the bond joining the nucleobase to the sugar, and nucleobase charge. The results suggest that stability of nucleobase pairs correlates with the no. of H-bonds, size complementarity, the presence of uncompensated functional groups, and the presence of charge on a nucleobase. Each of these properties appear to be more significant than the nature of the glycosidic bond and sequence context. The results provide guidelines for constructing stable Watson-Crick like nucleobase pairs with unnatural nucleobases. The expts. also demonstrate that expanded genetic systems can be constructed using size complementary nucleobase pairs that contain three hydrogen bonds.
5盖尔，C. R.， 巴特斯比， TR和 Benner， S. A. （2003） 扩展的 Watson-Crick 样遗传信息系统中的核碱基配对结构 11， 1485 DOI： 10.1016/j.str.2003.11.008Google Scholar内容扩展的 Watson-Crick 样遗传信息系统中的 cas_cit5R“>5 核碱基配对Geyer， C. Ronald;巴特斯比，托马斯 R.;本纳，史蒂文 A.结构（美国马萨诸塞州剑桥市） （2003 年）、 11 （12）， 1485 年至 1498 年科登： STRUE6; 国际标准书号：0969-2126。 （细胞出版社）为了指导替代遗传系统的设计，我们测量了来自天然和非自然结构的 DNA 双链体的匹配和不匹配核碱基对的熔解温度。 根据结构特征分析了这些对，包括核碱基大小，没有。形成的氢键、未补偿氢键官能团的存在、将核碱基连接到糖的键的性质以及核碱基电荷。 结果表明，核碱基对的稳定性与 no.H 键、尺寸互补性、未补偿官能团的存在以及核碱基上电荷的存在。 这些特性中的每一种似乎都比糖苷键和序列上下文的性质更重要。 结果为构建具有非天然核碱基的稳定 Watson-Crick 样核碱基对提供了指导。 外籍人士。还证明可以使用包含三个氢键的大小互补核碱基对构建扩展的遗传系统。https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXps1OrtL0%253D&md5=4fdcf5a3d34c73b118c90f57246cbc11 的更多 - 6Yakovchuk, P., Protozanova, E., and Frank-Kamenetskii, M. D. (2006) Base-stacking and base-pairing contributions into thermal stability of the DNA double helix Nucleic Acids Res. 34, 564 DOI: 10.1093/nar/gkj454Google Scholar 谷歌学术6https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XhtlOnuro%253D&md5=2c72728f600373744c60a81684a64409Base-stacking and base-pairing contributions into thermal stability of the DNA double helixYakovchuk, Peter; Protozanova, Ekaterina; Frank-Kamenetskii, Maxim D.Nucleic Acids Research (2006), 34 (2), 564-574CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)Two factors are mainly responsible for the stability of the DNA double helix: base pairing between complementary strands and stacking between adjacent bases. By studying DNA mols. with solitary nicks and gaps we measure temp. and salt dependence of the stacking free energy of the DNA double helix. For the first time, DNA stacking parameters are obtained directly (without extrapolation) for temps. from below room temp. to close to melting temp. We also obtain DNA stacking parameters for different salt concns. ranging from 15 to 100 mM Na+. From stacking parameters of individual contacts, we calc. base-stacking contribution to the stability of A•T- and G•C-contg. DNA polymers. We find that temp. and salt dependences of the stacking term fully det. the temp. and the salt dependence of DNA stability parameters. For all temps. and salt concns. employed in present study, base-stacking is the main stabilizing factor in the DNA double helix. A•T pairing is always destabilizing and G•C pairing contributes almost no stabilization. Base-stacking interaction dominates not only in the duplex overall stability but also significantly contributes into the dependence of the duplex stability on its sequence.
6雅科夫丘克，P.， 普罗托扎诺娃， E.和 Frank-Kamenetskii， M. D. （2006） 碱基堆叠和碱基配对对 DNA 双螺旋核酸热稳定性的贡献 Res. 34， 564 DOI： 10.1093/NAR/GKJ454Google Scholar内容6碱基堆叠和碱基配对对 DNA 双螺旋热稳定性的贡献Yakovchuk， Peter;Protozanova， 叶卡捷琳娜;Frank-Kamenetskii， 马克西姆 D.核酸研究 （2006 年）、 34 （2）， 564-574 号科登： 纳尔哈德; 国际标准书号：0305-1048。 （牛津大学出版社）两个因素主要负责 DNA 双螺旋的稳定性：互补链之间的碱基配对和相邻碱基之间的堆叠。 通过研究 DNA mols.通过孤立的缺口和间隙，我们测量 DNA 双螺旋堆积自由能的温度和盐依赖性。 首次直接获得温度的 DNA 堆积参数（无需外推）。从低于室温到接近熔化温度。 我们还获得了不同盐浓度的 DNA 堆积参数。范围为 15 至 100 mM Na+。 根据单个触点的堆叠参数，我们计算了底座堆叠对 A•T 和 G•C -contg 稳定性的贡献。DNA 聚合物。 我们发现，堆积项的温度和盐依赖性完全取决于 DNA 稳定性参数的温度和盐依赖性。 适用于所有温度。和盐 concns。在本研究中，碱基堆叠是 DNA 双螺旋中的主要稳定因子。 A•T 配对总是不稳定的，而 G•C 配对几乎没有稳定的作用。 碱基堆叠相互作用不仅在双链体整体稳定性中占主导地位，而且对双链体稳定性对其序列的依赖性也有显著贡献。 - 7Benner, S. A., Karalkar, N. B., Hoshika, S., Laos, R., Shaw, R. W., Matsuura, M., Fajardo, D., and Moussatche, P. (2016) Alternative Watson-Crick Synthetic Genetic Systems Cold Spring Harbor Perspect. Biol. 8, a023770 DOI: 10.1101/cshperspect.a023770Google Scholar 谷歌学术7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXpslWktrY%253D&md5=db3376ae594ae9894e2901926bf65133Alternative Watson-Crick synthetic genetic systemsBenner, Steven A.; Karalkar, Nilesh B.; Hoshika, Shuichi; Laos, Roberto; Shaw, Ryan W.; Matsuura, Mariko; Fajardo, Diego; Moussatche, PatriciaCold Spring Harbor Perspectives in Biology (2016), 8 (11), a023770/1-a023770/27CODEN: CSHPEU; ISSN:1943-0264. (Cold Spring Harbor Laboratory Press)In its "grand challenge" format in chem., "synthesis" as an activity sets out a goal that is substantially beyond current theor. and technol. capabilities. In pursuit of this goal, scientists are forced across uncharted territory, where they must answer unscripted questions and solve unscripted problems, creating new theories and new technologies in ways that would not be created by hypothesis-directed research. Thus, synthesis drives discoveryand paradigm changes in waysthat anal. cannot. Described here are the products that have arisen so far through the pursuit of one grand challenge in synthetic biol.: Recreate the genetics, catalysis, evolution, and adaptation that we value in life, but using genetic and catalytic biopolymers different from those that have been delivered to us by natural historyon Earth. The outcomes in technol. include new diagnostic toolsthat have helped personalize the care of hundreds of thousands of patients worldwide. In science, the effort has generated a fundamentally different view of DNA, RNA, and how they work.
7本纳，SA， 卡拉卡尔， NB， 星香， S.， 老挝， R.， 肖， RW， 松浦， M.， 法哈多， D.和 Moussatche， P. （2016） 替代 Watson-Crick 合成遗传系统冷泉港观察。生物学 8， a023770 DOI： 10.1101/cshperspect.a023770Google Scholar内容7替代 Watson-Crick 合成遗传系统Benner， Steven A.;卡拉卡尔，尼莱什 B.;星香，Shuichi;老挝，罗伯托;肖，瑞恩 W.;松浦麻理子;迭戈·法哈多;Moussatche， PatriciaCold Spring Harbor 生物学观点 （2016 年）、 8 （11）、 编号： A023770/1-A023770/27科登： CSHPEU; 国际标准书号：1943-0264。 （冷泉港实验室出版社）在化学的“大挑战”形式中，“合成”作为一种活动设定了一个大大超出当前理论和技术能力的目标。 为了实现这一目标，科学家们被迫跨越未知领域，他们必须回答无脚本的问题并解决无脚本的问题，以假设导向的研究无法创造的方式创造新理论和新技术。 因此，综合以 anal. 无法做到的方式推动发现和范式变化。 这里描述的是迄今为止通过追求合成生物学中的一项重大挑战而产生的产品：重建我们在生活中重视的遗传学、催化、进化和适应，但使用与地球上自然历史提供给我们的不同的遗传和催化生物聚合物。 技术成果包括新的诊断工具，这些工具已帮助全球数十万患者提供个性化护理。 在科学领域，这项工作产生了对 DNA、RNA 及其工作原理的根本不同的看法。 - 8Lutz, M. J., Horlacher, J., and Benner, S. A. (1998) Recognition of a non-standard base pair by thermostable DNA polymerases Bioorg. Med. Chem. Lett. 8, 1149 DOI: 10.1016/S0960-894X(98)00177-2
8卢茨，MJ， 霍拉赫， J.和 Benner， S. A. （1998） 热稳定 DNA 聚合酶对非标准碱基对的识别 Bioorg. Med. Chem. Lett. 8， 1149 DOI： 编号：10.1016/S0960-894X（98）00177-2 - 9Yang, Z., Chen, F., Chamberlin, S. G., and Benner, S. A. (2010) Expanded genetic alphabets in the polymerase chain reaction Angew. Chem., Int. Ed. 49, 177 DOI: 10.1002/anie.200905173Google Scholar 谷歌学术9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhs1Wlt7fP&md5=265d5ee46db9fd09fd0457ba6a471378Expanded genetic alphabets in the polymerase chain reactionYang, Zunyi; Chen, Fei; Chamberlin, Stephen G.; Benner, Steven A.Angewandte Chemie, International Edition (2010), 49 (1), 177-180, S177/1-S177/16CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)A flexible nested PCR using an artificially expanded genetic information system (AEGIS) was developed. A pyrimidine analog (Z) and its complementary purine analog (P) were used as extra genetic letters. Incorporated into DNA strands, they form Z:P pairs that contribute to duplex thermostability more than C:G pairs, discriminate against mismatches to the same extent as std. nucleobases, and are accepted by many polymerases, including Deep Vent and Taq.
9杨 Z.， 陈 F.， 张伯伦， SG和 Benner， S. A. （2010） 聚合酶链反应中的扩展遗传字母表。Angew. Chem.， Int. Ed. 49， 177 DOI： 10.1002/anie.200905173Google Scholar内容9聚合酶链反应中扩增的遗传字母Yang， 遵义;陈飞;张伯伦，斯蒂芬 G.;本纳，史蒂文 A.Angewandte Chemie，国际版 （2010 年）， 49 （1）、 177-180 号，S177/1-S177/16科登： ACIEF5; 国际标准书号：1433-7851。 （Wiley-VCH Verlag GmbH & Co. KGaA）开发了一种使用人工扩增遗传信息系统 （AEGIS） 的灵活巢式 PCR。 嘧啶类似物 （Z） 及其互补嘌呤类似物 （P） 用作额外的遗传字母。 它们掺入 DNA 链中，形成 Z：P 对，比 C：G 对更有助于双链体热稳定性，对错配的区分程度与标准核碱基相同，并被许多聚合酶接受，包括 Deep Vent 和 Taq。 - 10Yang, Z., Chen, F., Alvarado, J. B., and Benner, S. A. (2011) Amplification, mutation, and sequencing of a six-letter synthetic genetic system J. Am. Chem. Soc. 133, 15105 DOI: 10.1021/ja204910nGoogle Scholar 谷歌学术10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtFaks7zL&md5=cc4561e9527ca359c82494deb9e0be2dAmplification, mutation, and sequencing of a six-letter synthetic genetic systemYang, Zun-Yi; Chen, Fei; Alvarado, J. Brian; Benner, Steven A.Journal of the American Chemical Society (2011), 133 (38), 15105-15112CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The next goals in the development of a synthetic biol. that uses artificial genetic systems will require chem.-biol. combinations that allow the amplification of DNA contg. any no. of sequential and nonsequential nonstandard nucleotides. This amplification must ensure that the nonstandard nucleotides are not unidirectionally lost during PCR amplification (unidirectional loss would cause the artificial system to revert to an all-natural genetic system). Further, technol. is needed to sequence artificial genetic DNA mols. The work reported here meets all three of these goals for a six-letter artificially expanded genetic information system (AEGIS) that comprises four std. nucleotides (G, A, C, and T) and two addnl. nonstandard nucleotides (Z and P). We report polymerases and PCR conditions that amplify a wide range of GACTZP DNA sequences having multiple consecutive unnatural synthetic genetic components with low (0.2% per theor. cycle) levels of mutation. We demonstrate that residual mutation processes both introduce and remove unnatural nucleotides, allowing the artificial genetic system to evolve as such, rather than revert to a wholly natural system. We then show that mechanisms for these residual mutation processes can be exploited in a strategy to sequence "six-letter" GACTZP DNA. These are all not yet reported for any other synthetic genetic system.
10杨 Z.， 陈 F.， 阿尔瓦拉多， JB和 Benner， S. A. （2011） 六字母合成遗传系统的扩增、突变和测序 J. Am. Chem. Soc. 133， 15105 DOI： 10.1021/JA204910NGoogle Scholar内容10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtFaks7zL&md5=cc4561e9527ca359c82494deb9e0be2d 的更多一个六字母合成遗传系统的扩增、突变和测序Yang， Zun-Yi;陈飞;阿尔瓦拉多，J. 布莱恩;本纳，史蒂文 A.美国化学会杂志 （2011 年）， 133 元 （38）， 15105-15112科登： JACSAT公司; 国际标准书号：0002-7863。 （美国化学学会）开发使用人工遗传系统的合成生物学的下一个目标将需要化学-生物组合，这些组合允许扩增 DNA 连续和非连续非标准核苷酸的数量。 这种扩增必须确保非标准核苷酸在 PCR 扩增过程中不会单向丢失（单向丢失会导致人工系统恢复到全天然遗传系统）。 此外，需要技术来对人工遗传 DNA 分子进行测序。 这里报告的工作满足了六个字母的人工扩展遗传信息系统 （AEGIS） 的所有三个目标，该系统包括四个标准核苷酸（G、A、C 和 T）和两个附加的非标准核苷酸（Z 和 P）。 我们报道了扩增各种 GACTZP DNA 序列的聚合酶和 PCR 条件，这些序列具有多个连续的非天然合成遗传成分，突变水平低（每个理论循环 0.2%）。 我们证明，残余突变过程既会引入又会去除非天然核苷酸，从而允许人工遗传系统进化成这样，而不是恢复到完全自然的系统。 然后，我们表明这些残留突变过程的机制可以在对“六个字母”GACTZP DNA 进行测序的策略中加以利用。 这些都尚未针对任何其他合成遗传系统进行报道。 - 11Georgiadis, M. M., Singh, I., Kellett, W. F., Hoshika, S., Benner, S. A., and Richards, N. G. (2015) Structural basis for a six nucleotide genetic alphabet J. Am. Chem. Soc. 137, 6947 DOI: 10.1021/jacs.5b03482Google Scholar 谷歌学术11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXot1SqtLc%253D&md5=243dac24bcf9c2a3b8e72c822eac7c74Structural Basis for a Six Nucleotide Genetic AlphabetGeorgiadis, Millie M.; Singh, Isha; Kellett, Whitney F.; Hoshika, Shuichi; Benner, Steven A.; Richards, Nigel G. J.Journal of the American Chemical Society (2015), 137 (21), 6947-6955CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Expanded genetic systems are most likely to work with natural enzymes if the added nucleotides pair with geometries that are similar to those displayed by std. duplex DNA. Here, we present crystal structures of 16-mer duplexes showing this to be the case with two nonstandard nucleobases (Z, 6-amino-5-nitro-2(1H)-pyridone and P, 2-amino-imidazo[1,2-a]-1,3,5-triazin-4(8H)one) that were designed to form a Z:P pair with a std. "edge on" Watson-Crick geometry, but joined by rearranged hydrogen bond donor and acceptor groups. One duplex, with four Z:P pairs, was crystd. with a reverse transcriptase host and adopts primarily a B-form. Another contained six consecutive Z:P pairs; it crystd. without a host in an A-form. In both structures, Z:P pairs fit canonical nucleobase hydrogen-bonding parameters and known DNA helical forms. Unique features include stacking of the nitro group on Z with the adjacent nucleobase ring in the A-form duplex. In both B- and A-duplexes, major groove widths for the Z:P pairs are approx. 1 Å wider than those of comparable G:C pairs, perhaps to accommodate the large nitro group on Z. Otherwise, ZP-rich DNA had many of the same properties as CG-rich DNA, a conclusion supported by CD studies in soln. The ability of std. duplexes to accommodate multiple and consecutive Z:P pairs is consistent with the ability of natural polymerases to biosynthesize those pairs. This, in turn, implies that the GACTZP synthetic genetic system can explore the entire expanded sequence space that addnl. nucleotides create, a major step forward in this area of synthetic biol.
11乔治亚迪斯，M. M.， 辛格， I.， 凯利特， WF， 星香， S.， 本纳， SA和理查兹，N. G. （2015） 六核苷酸遗传字母的结构基础 J. Am. Chem. Soc. 137， 6947 DOI： 10.1021/jacs.5b03482Google Scholar11内容？origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXot1SqtLc%253D&md5=243dac24bcf9c2a3b8e72c822eac7c74六核苷酸遗传字母的结构基础Georgiadis， Millie M.;辛格，伊莎;凯利特，惠特尼 F.;星香，Shuichi;本纳，史蒂文 A.;理查兹，奈杰尔 GJ美国化学会杂志 （2015 年）， 票价 137 元 （21）、 6947-6955科登： JACSAT公司; 国际标准书号：0002-7863。 （美国化学学会）如果添加的核苷酸与与标准双链 DNA 显示的几何形状相似，则扩增的遗传系统最有可能与天然酶一起工作。 在这里，我们展示了 16 聚体双链体的晶体结构，表明这是两个非标准核碱基（Z，6-氨基-5-硝基-2（1H）-吡啶酮和 P，2-氨基-咪唑[1,2-a]-1,3,5-三嗪-4（8H）one）的情况，它们被设计为形成具有标准“边缘”的 Z：P 对 Watson-Crick 几何形状，但由重排的氢键供体和受体基团连接。 一个双工，有四个 Z：P 对，是 crystd。与逆转录酶宿主，主要采用 B 型。 另一个包含六个连续的 Z：P 对;它哭泣。没有 A 表单中的主机。 在这两种结构中，Z：P 对都符合规范核碱基氢键参数和已知的 DNA 螺旋形式。 独特的功能包括在 Z 上堆积硝基与 A 型双链体中相邻的核碱基环。 在 B 和 A 双工中，Z：P 对的大槽宽度比同类 G：C 对的大槽宽约 1 Å，可能是为了容纳 Z 上的大硝基。 否则，富含 ZP 的 DNA 具有许多与富含 CG 的 DNA 相同的特性，这一结论得到了 soln 的 CD 研究的支持。 标准双链体容纳多个和连续的 Z：P 对的能力与天然聚合酶生物合成这些对的能力一致。 反过来，这意味着 GACTZP 合成遗传系统可以探索 addnl. 核苷酸创造的整个扩展序列空间，这是合成生物学领域向前迈出的重要一步。 - 12Sismour, A. M., Lutz, S., Park, J. H., Lutz, M. J., Boyer, P. L., Hughes, S. H., and Benner, S. A. (2004) PCR amplification of DNA containing non-standard base pairs by variants of reverse transcriptase from Human Immunodeficiency Virus-1 Nucleic Acids Res. 32, 728 DOI: 10.1093/nar/gkh241
12西斯莫尔，上午， 卢茨， S.， 帕克， JH， 卢茨， MJ， 博耶， PL， 休斯， SH和 Benner， S. A. （2004） 通过人类免疫缺陷病毒 1 核酸逆转录酶变体对含有非标准碱基对的 DNA 进行 PCR 扩增 Res. 32， 728 DOI： 10.1093/纳尔/GKH241 - 13Winiger, C. B., Kim, M. J., Hoshika, S., Shaw, R. W., Moses, J. D., Matsuura, M. F., Gerloff, D. L., and Benner, S. A. (2016) Polymerase Interactions with Wobble Mismatches in Synthetic Genetic Systems and Their Evolutionary Implications Biochemistry 55, 3847 DOI: 10.1021/acs.biochem.6b00533Google Scholar 谷歌学术13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtVGjtL%252FO&md5=80d5724b34f1228c5b0450f17f5f56c1Polymerase Interactions with Wobble Mismatches in Synthetic Genetic Systems and Their Evolutionary ImplicationsWiniger, Christian B.; Kim, Myong-Jung; Hoshika, Shuichi; Shaw, Ryan W.; Moses, Jennifer D.; Matsuura, Mariko F.; Gerloff, Dietlind L.; Benner, Steven A.Biochemistry (2016), 55 (28), 3847-3850CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)In addn. to completing the Watson- Crick nucleobase matching "concept" (big pairs with small, hydrogen bond donors pair with hydrogen bond acceptors), artificially expanded genetic information systems (AEGIS) also challenge DNA polymerases with a complete set of mismatches, including wobble mismatches. Here, we explore wobble mismatches with AEGIS with DNA polymerase 1 from Escherichia coli. Remarkably, we find that the polymerase tolerates an AEGIS:std. wobble that has the same geometry as the G:T wobble that polymerases have evolved to exclude but excludes a wobble geometry that polymerases have never encountered in natural history. These results suggest certain limits to "structural analogy" and "evolutionary guidance" as tools to help synthetic biologists expand DNA alphabets.
13维尼格，CB， 金， MJ， 星香， S.， 肖， RW， 摩西， J. D.， 松浦， MF， 格洛夫， DL和 Benner， S. A. （2016） 合成遗传系统中聚合酶与摆动错配的相互作用及其进化意义，生物化学 55， 3847 DOI： 10.1021/acs.biochem.6b00533Google Scholar内容13合成遗传系统中聚合酶与摆动错配的相互作用及其进化意义Winiger， Christian B.;Kim， Myong-Jung;星香，Shuichi;肖，瑞恩 W.;摩西，詹妮弗 D.;松浦麻理子 F.;格洛夫，迪特林德 L.;本纳，史蒂文 A.生物化学 （2016 年）、 55 元 （28）、 3847-3850科登： 比肖; 国际标准书号：0006-2960。 （美国化学学会）除了完成 Watson-Crick 核碱基匹配“概念”（大对与小氢键供体与氢键受体配对）之外，人工扩展遗传信息系统 （AEGIS） 还以一整套错配（包括摆动错配）挑战 DNA 聚合酶。 在这里，我们探讨了 AEGIS 与来自大肠杆菌的 DNA 聚合酶 1 的摆动错配。 值得注意的是，我们发现聚合酶可以容忍 AEGIS：std. 摆动，该摆动与聚合酶进化以排除的 G：T 摆动具有相同的几何形状，但排除了聚合酶在自然史上从未遇到过的摆动几何形状。 这些结果表明，作为帮助合成生物学家扩展 DNA 字母表的工具，“结构类比”和“进化指导”存在一定的局限性。 - 14Winiger, C. B., Shaw, R. W., Kim, M. J., Moses, J. D., Matsuura, M. F., and Benner, S. A. (2017) Expanded Genetic Alphabets: Managing Nucleotides That Lack Tautomeric, Protonated, or Deprotonated Versions Complementary to Natural Nucleotides ACS Synth. Biol. 6, 194 DOI: 10.1021/acssynbio.6b00193Google Scholar 谷歌学术14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhsFakurvO&md5=820723e07e1274af995b78de5bed483aExpanded Genetic Alphabets: Managing Nucleotides That Lack Tautomeric, Protonated, or Deprotonated Versions Complementary to Natural NucleotidesWiniger, Christian B.; Shaw, Ryan W.; Kim, Myong-Jung; Moses, Jennifer D.; Matsuura, Mariko F.; Benner, Steven A.ACS Synthetic Biology (2017), 6 (2), 194-200CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)2,4-Diaminopyrimidine (trivially K) and imidazo[1,2-a]-1,3,5-triazine-2(8H)-4(3H)-dione (trivially X) form a nucleobase pair with Watson-Crick geometry as part of an artificially expanded genetic information system (AEGIS). Unlike some other AEGIS components, neither K nor X have any minor tautomeric forms mispairing. In vivo and in vitro expts. show how DNA polymerase I from E. coli manages this geometric distortion in DNA with one K:X pair, and fails to manage it with two adjacent K:X pairs. In the analogous in vivo expt., E. coli lacking dKTP/dXTP cannot rescue chloramphenicol resistance from a plasmid contg. two adjacent K:X pairs. In addn. to illustrating how living bacteria manage unnatural nucleotides, these studies identify bacteria able to serve as selection environments for engineering cells that replicate AEGIS pairs.or any protonated-deprotonated states that are Watson-Crick complementary to any natural nucleobase. Thus, K:X pairs can be replaced by natural pairs during replication only by geometrically distorted base pairing that is not tolerated by either the polymerase or its repair mechanisms.
14维尼格，CB， 肖， RW， 金， MJ， 摩西， J. D.， 松浦， MF和 Benner， S. A. （2017） 扩展遗传字母表：管理缺乏与天然核苷酸互补的互变异体、质子化或去质子化版本的核苷酸 ACS Synth. Biol. 6， 194 DOI： 10.1021/acssynbio.6b00193Google Scholar内容14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhsFakurvO&md5=820723e07e1274af995b78de5bed483a 的更多扩展遗传字母表：管理缺乏与天然核苷酸互补的互变异母体、质子化或去质子化版本的核苷酸Winiger， Christian B.;肖，瑞恩 W.;Kim， Myong-Jung;摩西，詹妮弗 D.;松浦麻理子 F.;本纳，史蒂文 A.ACS 合成生物学 （2017 年）， 6 （2）， 194-200 元科登： ASBCD6; 国际标准书号：2161-5063。 （美国化学学会）2,4-二氨基嘧啶（三重 K）和咪唑[1,2-a]-1,3,5-三嗪-2（8H）-4（3H）-二酮（三重 X）形成具有 Watson-Crick 几何学的核碱基对，作为人工扩展遗传信息系统 （AEGIS） 的一部分。 与其他一些 AEGIS 组件不同，K 和 X 都没有任何次要的互变异构形式错配。 体内和体外实验。显示了来自大肠杆菌的 DNA 聚合酶 I 如何用一个 K：X 对来管理 DNA 中的这种几何扭曲，而用两个相邻的 K：X 对则无法控制它。 在类似的体内实验中，缺乏 dKTP/dXTP 的大肠杆菌不能从质粒浓度中拯救氯霉素耐药性。两个相邻的 K：X 对。 为了说明活细菌如何管理非天然核苷酸，这些研究确定了能够作为复制 AEGIS 对的工程细胞的选择环境的细菌，或任何与任何天然核碱基互补的质子化-去质子化状态。 因此，K：X 对在复制过程中只能通过聚合酶或其修复机制不能容忍的几何扭曲碱基配对被天然对取代。 - 15Benner, S. A. and Shaw, R. W. In Vivo Conversion of Nucleosides in Plasmid DNA. US Patent Application 14218405, 2015.Google Scholar 谷歌学术There is no corresponding record for this reference.
15Benner， SA 和 Shaw， RW质粒 DNA 中核苷的体内转化。美国专利申请 14218405,2015。 - 16Betz, K., Malyshev, D. A., Lavergne, T., Welte, W., Diederichs, K., Dwyer, T. J., Ordoukhanian, P., Romesberg, F. E., and Marx, A. (2012) KlenTaq polymerase replicates unnatural base pairs by inducing a Watson-Crick geometry Nat. Chem. Biol. 8, 612 DOI: 10.1038/nchembio.966Google Scholar 谷歌学术16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XnvVyrurw%253D&md5=3f53105607a419a1d1326125178d682dKlenTaq polymerase replicates unnatural base pairs by inducing a Watson-Crick geometryBetz, Karin; Malyshev, Denis A.; Lavergne, Thomas; Welte, Wolfram; Diederichs, Kay; Dwyer, Tammy J.; Ordoukhanian, Phillip; Romesberg, Floyd E.; Marx, AndreasNature Chemical Biology (2012), 8 (7), 612-614CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)Many candidate unnatural DNA base pairs have been developed, but some of the best-replicated pairs adopt intercalated structures in free DNA that are difficult to reconcile with known mechanisms of polymerase recognition. Here we present crystal structures of KlenTaq DNA polymerase at different stages of replication for one such pair, dNaM-d5SICS, and show that efficient replication results from the polymerase itself, inducing the required natural-like structure.
16贝茨，K.， 马雷舍夫， DA， 拉弗涅， T.， 韦尔特， W.， 迪德里希斯， K.， 德怀尔， TJ， Ordoukhanian， P.， 罗梅斯伯格， FE和 Marx， A. （2012） KlenTaq 聚合酶通过诱导 Watson-Crick 几何来复制非自然碱基对 Nat. Chem. Biol. 8， 612 DOI： 10.1038/nchembio.966Google Scholar内容16KlenTaq 聚合酶通过诱导 Watson-Crick 几何形状来复制非自然碱基对Betz，Karin;马雷舍夫，丹尼斯 A.;拉弗涅，托马斯;韦尔特，沃尔夫拉姆;迪德里希斯，凯;德怀尔，塔米 J.;奥尔杜哈尼安，菲利普;罗梅斯伯格，弗洛伊德 E.;Marx， AndreasNature 化学生物学 （2012 年）、 8 （7）、 612-614科登： NCBABT 的; 国际标准书号：1552-4450。 （自然出版集团）已经开发了许多候选非天然 DNA 碱基对，但一些最佳复制对采用游离 DNA 中的嵌入结构，这很难与已知的聚合酶识别机制相协调。 在这里，我们展示了 KlenTaq DNA 聚合酶在不同复制阶段的晶体结构，即 dNaM-d5SICS，并表明聚合酶本身会产生有效的复制结果，从而诱导所需的天然样结构。 - 17Malyshev, D. A., Dhami, K., Lavergne, T., Chen, T., Dai, N., Foster, J. M., Correa, I. R., Jr., and Romesberg, F. E. (2014) A semi-synthetic organism with an expanded genetic alphabet Nature 509, 385 DOI: 10.1038/nature13314Google Scholar 谷歌学术17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXotVyqtb8%253D&md5=97b4b184cda52cc809b1705e5e88ad8eA semi-synthetic organism with an expanded genetic alphabetMalyshev, Denis A.; Dhami, Kirandeep; Lavergne, Thomas; Chen, Tingjian; Dai, Nan; Foster, Jeremy M.; Correa, Ivan R.; Romesberg, Floyd E.Nature (London, United Kingdom) (2014), 509 (7500), 385-388CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Organisms are defined by the information encoded in their genomes, and since the origin of life this information has been encoded using a two-base-pair genetic alphabet (A-T and G-C). In vitro, the alphabet has been expanded to include several unnatural base pairs (UBPs). We have developed a class of UBPs formed between nucleotides bearing hydrophobic nucleobases, exemplified by the pair formed between d5SICS and dNaM (d5SICS-dNaM), which is efficiently PCR-amplified and transcribed in vitro, and whose unique mechanism of replication has been characterized. However, expansion of an organism's genetic alphabet presents new and unprecedented challenges: the unnatural nucleoside triphosphates must be available inside the cell; endogenous polymerases must be able to use the unnatural triphosphates to faithfully replicate DNA contg. the UBP within the complex cellular milieu; and finally, the UBP must be stable in the presence of pathways that maintain the integrity of DNA. Here we show that an exogenously expressed algal nucleotide triphosphate transporter efficiently imports the triphosphates of both d5SICS and dNaM (d5SICSTP and dNaMTP) into Escherichia coli, and that the endogenous replication machinery uses them to accurately replicate a plasmid contg. d5SICS-dNaM. Neither the presence of the unnatural triphosphates nor the replication of the UBP introduces a notable growth burden. Lastly, we find that the UBP is not efficiently excised by DNA repair pathways. Thus, the resulting bacterium is the first organism to propagate stably an expanded genetic alphabet.
17马雷舍夫，DA， 达米， K.， 拉弗涅， T.， 陈 T.， 戴， N.， 福斯特， JM， 科雷亚， IR， Jr.和 Romesberg， F. E. （2014） 具有扩展遗传字母表的半合成生物体 Nature 509， 385 DOI： 10.1038/自然13314Google Scholar17内容？origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXotVyqtb8%253D&md5=97b4b184cda52cc809b1705e5e88ad8e一种具有扩展遗传字母表的半合成生物体Malyshev， Denis A.;达米，基兰迪普;拉弗涅，托马斯;陈廷健;戴楠;福斯特，杰里米 M.;科雷亚，伊万 R.;罗梅斯伯格，弗洛伊德 E.Nature （英国 伦敦） （2014 年）、 509 元 （7500）、 385-388 元科登： 纳图阿斯; 国际标准书号：0028-0836。 （自然出版集团）生物体是由其基因组中编码的信息定义的，自生命起源以来，这些信息一直使用两个碱基对的遗传字母表（A-T 和 G-C）进行编码。 在体外，字母表已扩展到包括几个非天然碱基对 （UBP）。 我们开发了一类在带有疏水核碱基的核苷酸之间形成的 UBP，以 d5SICS 和 dNaM 之间形成的一对 （d5SICS-dNaM） 为例，它在体外被有效地 PCR 扩增和转录，并且其独特的复制机制已被表征。 然而，生物体遗传字母表的扩展带来了前所未有的新挑战：非天然的核苷三磷酸盐必须在细胞内可用;内源性聚合酶必须能够使用非天然的三磷酸盐来忠实地复制 DNA contg。复杂细胞环境中的 UBP;最后，UBP 必须在存在维持 DNA 完整性的通路的情况下保持稳定。 在这里，我们表明外源表达的藻类核苷酸三磷酸转运蛋白有效地将 d5SICS 和 dNaM 的三磷酸盐（d5SICSTP 和 dNaMTP）输入到大肠杆菌中，并且内源性复制机制使用它们来准确复制质粒浓度。d5SICS-dNaM 的。 非天然三磷酸盐的存在和 UBP 的复制都没有带来显著的生长负担。 最后，我们发现 UBP 不能被 DNA 修复途径有效切除。 因此，所得细菌是第一个稳定繁殖扩展的遗传字母表的生物体。 - 18Neidle, S. (1999) Nucleic Acid Structure, Oxford University Press, New York.Google Scholar 谷歌学术There is no corresponding record for this reference.
18Neidle， S. （1999） 核酸结构，牛津大学出版社，纽约。 - 19Molt, R. W., Jr., Georgiadis, M. M., and Richards, N. G. J. (2017) Consecutive non-natural PZ nucleobase pairs in DNA impact helical structure as seen in 50 mus molecular dynamics simulations Nucleic Acids Res. 45, 3643 DOI: 10.1093/nar/gkx144
19小莫尔特， 乔治亚迪斯， MM。和理查兹，新墨西哥州 （2017） DNA 中连续的非天然 PZ 核碱基对影响螺旋结构，如 50 mus 分子动力学模拟中所见 核酸研究 45， 3643 DOI： 10.1093/纳尔/GKX144 - 20Sun, D., Jessen, S., Liu, C., Liu, X., Najmudin, S., and Georgiadis, M. M. (1998) Cloning, expression, and purification of a catalytic fragment of Moloney murine leukemia virus reverse transcriptase: crystallization of nucleic acid complexes Protein Sci. 7, 1575 DOI: 10.1002/pro.5560070711Google Scholar 谷歌学术20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXks1eqtLg%253D&md5=9e72f0850f5e4777c85e81a3a32f3074Cloning, expression, and purification of a catalytic fragment of Moloney murine leukemia virus reverse transcriptase: crystallization of nucleic acid complexesSun, Dunming; Jessen, Sven; Liu, Chunhui; Liu, Xiuping; Najmudin, Shabir; Georgiadis, Millie M.Protein Science (1998), 7 (7), 1575-1582CODEN: PRCIEI; ISSN:0961-8368. (Cambridge University Press)Reverse transcriptase is an essential retroviral enzyme that uses RNA- and DNA-directed DNA polymerase activities as well as an RNaseH activity to synthesize a double-stranded DNA copy of the single-stranded RNA genome. In an effort to obtain high-resoln. structural information regarding the polymerase active site of reverse transcriptase, we have pursued studies on a catalytic fragment from Moloney murine leukemia virus reverse transcriptase. DNA encoding the catalytic fragment, defined originally by limited proteolytic digestion, has been cloned, and the protein has been expressed and purified from Escherichia coli. The fragment obtained by limited proteolytic digestion and the bacterially expressed fragment retain polymerase activity. Crystn. studies involving nucleic acid complexes with a catalytic fragment from both sources are reported, including variables screened to improve crystals and cryocooling. Three crystal forms of catalytic fragment-nucleic acid complexes have been characterized, which all contain at least two protein mols. in the asym. unit. As isolated, the catalytic fragment is monomeric. This anal. indicates that the enzyme dimerizes in the presence of nucleic acid.
20Sun， D.， 杰森， S.， 刘 C.， 刘， X.， 纳吉穆丁， S.和 Georgiadis， M. M. （1998） 莫洛尼鼠白血病病毒逆转录酶催化片段的克隆、表达和纯化：核酸复合物的结晶 蛋白质科学 7， 1575 DOI： 10.1002/专业版 5560070711Google Scholar内容20莫洛尼鼠白血病病毒逆转录酶催化片段的克隆、表达和纯化：核酸复合物的结晶Sun， Dunming;杰森，斯文;刘春晖;刘秀平;纳吉穆丁，沙比尔;乔治亚迪斯，米莉 M.蛋白质科学 （1998 年）、 7 （7）、 1575-1582 年科登： PRCIEI; 国际标准书号：0961-8368。 （剑桥大学出版社）逆转录酶是一种必需的逆转录病毒酶，它使用 RNA 和 DNA 导向的 DNA 聚合酶活性以及 RNaseH 活性来合成单链 RNA 基因组的双链 DNA 拷贝。 为了获得高分辨率。关于逆转录酶聚合酶活性位点的结构信息，我们对莫洛尼鼠白血病病毒逆转录酶的催化片段进行了研究。 编码催化片段的 DNA（最初由有限的蛋白水解消化定义）已被克隆，并且该蛋白质已从大肠杆菌中表达和纯化。 通过有限蛋白水解消化获得的片段和细菌表达的片段保留了聚合酶活性。 克里斯特。报告了涉及核酸复合物与来自两种来源的催化片段的研究，包括筛选以改善晶体和低温冷却的变量。 已经表征了催化片段-核酸复合物的三种晶型，它们都含有至少两个蛋白质 mols。在 asym.单位。 分离时，催化片段是单体的。 该肛门表明酶在核酸存在下二聚化。 - 21Cote, M. L., Yohannan, S. J., and Georgiadis, M. M. (2000) Use of an N-terminal fragment from moloney murine leukemia virus reverse transcriptase to facilitate crystallization and analysis of a pseudo-16-mer DNA molecule containing G-A mispairs Acta Crystallogr., Sect. D: Biol. Crystallogr. 56, 1120 DOI: 10.1107/S0907444900008246
21科特，M. L.， 约翰南， SJ和 Georgiadis， M. M. （2000） 使用来自 moloney 鼠白血病病毒逆转录酶的 N 端片段促进含有 G-A 错配的伪 16 聚体 DNA 分子的结晶和分析Acta Crystallogr.， Sect. D： Biol. Crystallogr. 56， 1120 DOI： 10.1107/S0907444900008246 - 22Najmudin, S., Cote, M. L., Sun, D., Yohannan, S., Montano, S. P., Gu, J., and Georgiadis, M. M. (2000) Crystal structures of an N-terminal fragment from Moloney murine leukemia virus reverse transcriptase complexed with nucleic acid: functional implications for template-primer binding to the fingers domain J. Mol. Biol. 296, 613 DOI: 10.1006/jmbi.1999.3477
22纳吉穆丁， 科特， ML。， 孙， D.， 约翰南， S.， 蒙塔诺， SP， 顾 J.和 Georgiadis， M. M. （2000） 来自 Moloney 鼠白血病病毒逆转录酶与核酸复合的 N 端片段的晶体结构：模板引物结合到手指结构域的功能意义 J. Mol. Biol. 296， 613 DOI： 10.1006/jmbi.1999.3477 - 23Cote, M. L. and Georgiadis, M. M. (2001) Structure of a pseudo-16-mer DNA with stacked guanines and two G-A mispairs complexed with the N-terminal fragment of Moloney murine leukemia virus reverse transcriptase Acta Crystallogr., Sect. D: Biol. Crystallogr. 57, 1238 DOI: 10.1107/S090744490100943X
23科特，M. L. 和 Georgiadis， M. M. （2001） 具有堆叠鸟嘌呤和两个 G-A 错对的假 16-mer DNA 的结构，与莫洛尼鼠白血病病毒逆转录酶的 N 端片段复合，晶体学报，D 部分：生物学晶体学家。57， 1238 DOI： 10.1107/S090744490100943X - 24Montano, S. P., Cote, M. L., Roth, M. J., and Georgiadis, M. M. (2006) Crystal structures of oligonucleotides including the integrase processing site of the Moloney murine leukemia virus Nucleic Acids Res. 34, 5353 DOI: 10.1093/nar/gkl693
24蒙塔诺，SP， 科特， ML。， 罗斯， MJ和 Georgiadis， M. M. （2006） 寡核苷酸的晶体结构，包括莫洛尼鼠白血病病毒的整合酶加工位点 核酸研究 34， 5353 DOI： 10.1093 / 纳尔 / GKL693 - 25Joyce, C. M. and Steitz, T. A. (1994) Function and structure relationships in DNA polymerases Annu. Rev. Biochem. 63, 777 DOI: 10.1146/annurev.bi.63.070194.004021Google Scholar 谷歌学术25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2MXitlCgtg%253D%253D&md5=5980e61ddb57aa9d5e0ad27a7c3aab61Function and structure relationships in DNA polymerasesJoyce, Catherine M.; Steitz, Thomas A.Annual Review of Biochemistry (1994), 63 (), 777-822CODEN: ARBOAW; ISSN:0066-4154.A review, with 184 refs., on the structural and mechanistic aspects of the reactions catalyzed by DNA polymerase.
25乔伊斯，CM 和 Steitz， TA （1994） DNA 聚合酶的功能和结构关系 Annu. Rev. Biochem. 63， 777 DOI： 10.1146/annurev.bi.63.070194.004021Google Scholar的更多内容25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2MXitlCgtg%253D%253D&md5=5980e61ddb57aa9d5e0ad27a7c3aab61DNA 聚合酶中的功能和结构关系Joyce， Catherine M.;施泰茨，托马斯 A.生物化学年鉴 （1994 年）、 63 （）， 777-822科登： ARBOAW; 国际标准书号：0066-4154。一篇关于 DNA 聚合酶催化反应的结构和机制方面的综述，有 184 篇参考文献。 - 26Hendrickson, C. L., Devine, K. G., and Benner, S. A. (2004) Probing minor groove recognition contacts by DNA polymerases and reverse transcriptases using 3-deaza-2′-deoxyadenosine Nucleic Acids Res. 32, 2241 DOI: 10.1093/nar/gkh542
26亨德里克森，CL， 德文， K. G.和 Benner， S. A. （2004） 使用 3-deaza-2′-deoxyadenosine Nucleic Acids 探测 DNA 聚合酶和逆转录酶的小沟识别接触 Res. 32， 2241 DOI： 10.1093/纳尔/GKH542 - 27El Hassan, M. a. and Calladine, C. R. (1998) Two distinct modes of protein-induced bending in DNA J. Mol. Biol. 282, 331 DOI: 10.1006/jmbi.1998.1994Google Scholar 谷歌学术27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXmtlClsLc%253D&md5=f75d6fa540cc92d868bb129c3d110e22Two distinct modes of protein-induced bending in DNAEl Hassan, M. A.; Calladine, C. R.Journal of Molecular Biology (1998), 282 (2), 331-343CODEN: JMOBAK; ISSN:0022-2836. (Academic Press)Crystd. "naked" DNA oligomers in the B form show significant conformational mobility, particularly at CA/TG and TA/TA steps: there is a range in Roll angle of some 15° between consecutive base-pairs, and Slide and Twist are directly coupled to Roll. We call such motions "mode I". They are sufficient to enable DNA to curve gently around proteins such as histone octamers in the nucleosome particle. When DNA bends around other proteins, such as CAP and TBP, its distortion is much more severe. Although the DNA in close contact with these proteins includes the CA/TG and TA/TA steps, resp., the mode I flexibility is not deployed: instead, a more severe "mode II" manoeuvre is obsd. in DNA/protein co-crystals. Mode II has several distinctive phys. features. First, its range of Roll angle is much wider than for mode I. Second, the major-groove width remains more-or-less const. as Roll increases, whereas it decreases significantly as Roll increases in mode I; and this enables the major groove of the DNA to accommodate a protein moiety in its severely bent conformation. Third, the value of Slide remains more-or-less const. as Roll increases, whereas it decreases in mode I. In general, in both modes I and II, the major-groove width appears to be closely related to the Slide between base-pairs. In mode II there appears to be a definite "point pivot" on the major-groove side of the two base-pairs that constitute a dinucleotide step, formed either by the steric interlocking of propeller-twisted base-pairs or by a bifurcated hydrogen bond. Distortion of DNA in mode II seems to be an intrinsic property of the double-helical structure, since it occurs whether protein is bound on the major-groove side (e.g. CAP) or on the minor-groove side (e.g. TBP). Mode II distortion occurs in a wider range of steps than those that show the largest mode-I variation; nevertheless, "access" to mode II deformation appears to be gained via mode I distortion at particular steps CA/TG and TA/TA. (c) 1998 Academic Press.
27马萨诸塞州埃尔哈桑 和 Calladine， C. R. （1998） DNA 中蛋白质诱导弯曲的两种不同模式。J. Mol. Biol. 282， 331 DOI： 1998 年 10 月 1006 日/JMBI.1994Google Scholar27内容 https://chemport.cas.org/services/resolver？origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXmtlClsLc%253D&md5=f75d6fa540cc92d868bb129c3d110e22DNA 中蛋白质诱导弯曲的两种不同模式El Hassan， MA;卡拉丁，CR分子生物学杂志 （1998 年）、 282 元 （2）， 331-343 元科登： JMOBAK; 国际标准书号：0022-2836。 （学术出版社）Crystd.B 型的“裸”DNA 寡聚体显示出显著的构象迁移率，特别是在 CA/TG 和 TA/TA 步骤中：连续碱基对之间的 Roll 角范围约为 15°，并且 Slide 和 Twist 直接与 Roll 偶联。 我们将这种运动称为 “模式 I”。 它们足以使 DNA 在核小体颗粒中的蛋白质（如组蛋白八聚体）周围轻轻弯曲。 当 DNA 围绕其他蛋白质（如 CAP 和 TBP）弯曲时，其扭曲要严重得多。 尽管与这些蛋白质密切接触的 DNA 包括 CA/TG 和 TA/TA 步骤，但并没有部署 I 模式灵活性：相反，更严重的“模式 II”操作是 obsd。在 DNA/蛋白质共晶体中。 模式 II 有几个独特的物理特征。 首先，它的 Roll angle 范围比模式 I 宽得多。 其次，随着 Roll 的增加，大槽宽度或多或少保持不变，而在模式 I 中，随着 Roll 的增加，大槽宽度显着减小;这使得 DNA 的主要沟能够在其严重弯曲的构象中容纳蛋白质部分。 第三，随着 Roll 的增加，Slide 的值或多或少保持不变，而在模式 I 中则减小。 一般来说，在模式 I 和 II 中，大槽宽度似乎与基对之间的 Slide 密切相关。 在模式 II 中，在构成二核苷酸台阶的两个碱基对的大槽侧似乎有一个明确的“点枢轴”，由螺旋桨扭曲的碱基对的空间互锁或分叉的氢键形成。 模式 II 中 DNA 的扭曲似乎是双螺旋结构的内在特性，因为无论蛋白质是结合在大沟侧（例如 CAP）还是小沟侧（例如 TBP），都会发生这种扭曲。 模式 II 失真的发生范围比显示最大模式 I 变化的音阶范围更广;然而，对模式 II 变形的“访问”似乎是通过特定步骤 CA/TG 和 TA/TA 的模式 I 变形获得的。 （c） 1998 年学术出版社。 - 28Lu, X. J. and Olson, W. K. (2003) 3DNA: a software package for the analysis, rebuilding and visualization of three-dimensional nucleic acid structures Nucleic Acids Res. 31, 5108 DOI: 10.1093/nar/gkg680Google Scholar 谷歌学术28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXmvVWrsLo%253D&md5=959e6f79a9a71e0ca7269c2736e6a4c33DNA: a software package for the analysis, rebuilding and visualization of three-dimensional nucleic acid structuresLu, Xiang-Jun; Olson, Wilma K.Nucleic Acids Research (2003), 31 (17), 5108-5121CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)We present a comprehensive software package, 3DNA, for the anal., reconstruction and visualization of three-dimensional nucleic acid structures. Starting from a coordinate file in Protein Data Bank (PDB) format, 3DNA can handle antiparallel and parallel double helixes, single-stranded structures, triplexes, quadruplexes and other complex tertiary folding motifs found in both DNA and RNA structures. The anal. routines identify and categorize all base interactions and classify the double helical character of appropriate base pair steps. The program makes use of a recently recommended ref. frame for the description of nucleic acid base pair geometry and a rigorous matrix-based scheme to calc. local conformational parameters and rebuild the structure from these parameters. The rebuilding routines produce rectangular block representations of nucleic acids as well as full at. models with the sugar-phosphate backbone and publication quality standardized' base stacking diagrams. Utilities are provided to locate the base pairs and helical regions in a structure and to reorient structures for effective visualization. Regular helical models based on x-ray diffraction measurements of various repeating sequences can also be generated within the program.
28卢 X. J. 和奥尔森，WK （2003） 3DNA：用于分析、重建和可视化三维核酸结构的软件包 核酸研究 31， 5108 DOI： 10.1093/纳尔/GKG680Google Scholar内容28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXmvVWrsLo%253D&md5=959e6f79a9a71e0ca7269c2736e6a4c3 的更多3DNA：用于三维核酸结构分析、重建和可视化的软件包卢祥军;奥尔森，威尔玛 K.核酸研究 （2003 年）、 31 （17）， 5108-5121科登： 纳尔哈德; 国际标准书号：0305-1048。 （牛津大学出版社）我们提出了一个全面的软件包 3DNA，用于肛门、三维核酸结构的重建和可视化。 从蛋白质数据库 （PDB） 格式的坐标文件开始，3DNA 可以处理 DNA 和 RNA 结构中的反平行和平行双螺旋、单链结构、三链、四链和其他复杂的三级折叠基序。 分析例程识别并分类所有碱基相互作用，并对适当碱基对步骤的双螺旋特征进行分类。 该程序利用最近推荐的参考框架来描述核酸碱基对的几何形状，并利用严格的基于基质的方案来计算局部构象参数并根据这些参数重建结构。 重建例程生成核酸的矩形块表示以及完整的 at。具有糖磷酸骨架和出版物质量标准化的模型“基础堆叠图。 提供了一些实用程序来定位结构中的碱基对和螺旋区域，并重新定位结构以实现有效的可视化。 还可以在程序中生成基于各种重复序列的 X 射线衍射测量的规则螺旋模型。 - 29Lu, X. J. and Olson, W. K. (2008) 3DNA: a versatile, integrated software system for the analysis, rebuilding and visualization of three-dimensional nucleic-acid structures Nat. Protoc. 3, 1213 DOI: 10.1038/nprot.2008.104Google Scholar 谷歌学术29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXotFaktbY%253D&md5=47da876b232ab75aaa6bda05fb6f10713DNA: a versatile, integrated software system for the analysis, rebuilding and visualization of three-dimensional nucleic-acid structuresLu, Xiang-Jun; Olson, Wilma K.Nature Protocols (2008), 3 (7), 1213-1227CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)The authors present a set of protocols showing how to use the 3DNA suite of programs to analyze, rebuild and visualize three-dimensional nucleic-acid structures. The software dets. a wide range of conformational parameters, including the identities and rigid-body parameters of interacting bases and base-pair steps, the nucleotides comprising helical fragments, the area of overlap of stacked bases and so on. The reconstruction of three-dimensional structure takes advantage of rigorously defined rigid-body parameters, producing rectangular block representations of the nucleic-acid bases and base pairs and all-atom models with approx. sugar-phosphate backbones. The visualization components create vector-based drawings and scenes that can be rendered as raster-graphics images, allowing for easy generation of publication-quality figures. The utility programs use geometric variables to control the view and scale of an object, for comparison of related structures. The commands run in seconds even for large structures. The software and related information are available at.
29卢 X. J. 和奥尔森，WK （2008） 3DNA：用于分析、重建和可视化三维核酸结构的多功能集成软件系统 Nat. Protoc. 3， 1213 DOI： 10.1038/nprot.2008.104Google Scholar内容293DNA：用于三维核酸结构分析、重建和可视化的多功能集成软件系统Lu， Xiang-Jun;奥尔森，威尔玛 K.Nature 实验方案 （2008 年）、 3 （7）、 1213-1227 年科登： NPARDW; 国际标准书号：1750-2799。 （自然出版集团）作者提出了一套方案，展示了如何使用 3DNA 程序套件来分析、重建和可视化三维核酸结构。 软件 dets.广泛的构象参数，包括相互作用碱基和碱基对步骤的身份和刚体参数、包含螺旋片段的核苷酸、堆叠碱基的重叠面积等。 三维结构的重建利用了严格定义的刚体参数，生成核酸碱基和碱基对的矩形块表示以及具有大约糖磷酸骨架的全原子模型。 可视化组件可创建基于矢量的绘图和场景，这些绘图和场景可以渲染为光栅图形图像，从而可以轻松生成出版质量的图形。 实用程序使用几何变量来控制对象的视图和比例，以便比较相关结构。 即使对于大型结构，命令也能在几秒钟内运行。 该软件和相关信息可在以下网址获得。 - 31Prive, G. G., Heinemann, U., Chandrasegaran, S., Kan, L. S., Kopka, M. L., and Dickerson, R. E. (1987) Helix geometry, hydration, and G.A mismatch in a B-DNA decamer Science 238, 498 DOI: 10.1126/science.3310237
31普里夫，G. G.， 海涅曼， U.， 钱德拉塞加兰， S.， 堪萨斯， L. S.， 科普卡， ML。和迪克森，R. E. （1987） B-DNA 十聚体科学中的螺旋几何形状、水合和 G.A 错配 238， 498 DOI： 10.1126/科学.3310237 - 32Grzeskowiak, K. (1996) Sequence-dependent structural variation in B-DNA Chem. Biol. 3, 785 DOI: 10.1016/S1074-5521(96)90062-9Google Scholar 谷歌学术32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28Xmslyjtb8%253D&md5=cbaa4eb2c36acbed548746dac2b0d583Sequence-dependent structural variation in B-DNAGrzeskowiak, KazimierzChemistry & Biology (1996), 3 (10), 785-790CODEN: CBOLE2; ISSN:1074-5521. (Current Biology)A review and discussion with 38 refs. Although fiber diffraction originally led to the belief that the structure of DNA would be a simple regular helix, x-ray crystallog. of synthetic oligomers has shown that both deformability and structure depend on sequence. However, the rules that det. these factors remain mysterious.
32格热斯科维亚克，K. （1996） B-DNA 化学生物学中的序列依赖性结构变异 Biol. 3， 785 DOI： 10.1016/S1074-5521（96）90062-9Google Scholar内容32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28Xmslyjtb8%253D&md5=cbaa4eb2c36acbed548746dac2b0d583 的更多B-DNA中的序列依赖性结构变异Grzeskowiak， Kazimierz化学与生物学 （1996 年）、 3 （10）、 785-790科登： CBOLE2; 国际标准书号：1074-5521。 （当代生物学）与 38 个参考文献的评论和讨论。 尽管纤维衍射最初导致人们相信 DNA 的结构将是一个简单的规则螺旋，但 X 射线晶体学。的合成低聚物已经表明，变形性和结构都取决于序列。 然而，消除这些因素的规则仍然是个谜。 - 34Drew, H. R., Wing, R. M., Takano, T., Broka, C., Tanaka, S., Itakura, K., and Dickerson, R. E. (1981) Structure of a B-DNA dodecamer: conformation and dynamics Proc. Natl. Acad. Sci. U. S. A. 78, 2179 DOI: 10.1073/pnas.78.4.2179Google Scholar 谷歌学术34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL3MXktVajt74%253D&md5=1278977a90de8b049e2577294d569c45Structure of a B-DNA dodecamer. I. Conformation and dynamicsDrew, Horace R.; Wing, Richard M.; Takano, Tsunehiro; Broka, Christopher; Tanaka, Shoji; Itakura, Keiichi; Dickerson, Richard E.Proceedings of the National Academy of Sciences of the United States of America (1981), 78 (4), 2179-83CODEN: PNASA6; ISSN:0027-8424.The crystal structure of the synthetic DNA dodecamer d(CpGpCpGpApApTpTpCpGpCpG) was refined to a residual error of R = 17.8% at 1.9-Å resoln. (2-σ data). The mol. forms slightly >1 complete turn of right-handed double-stranded B helix. The 2 ends of the helix overlap and interlock minor grooves with neighboring mols. up and down a 21 screw axis, producing a 19° bend in helix axis over the 11-base-pair steps of the dodecamer. In the center of the mol., where perturbation is least, the helix has a mean rotation of 36.9° per step, or 9.8 base pairs per turn. The mean propeller twist (total dihedral angle between base planes) between A·T base pairs in the center of the mol. is 17.3°, and that between C·G pairs on the 2 ends avs. 11.5°. Individual deoxyribose ring conformations, measured by the C5'-C4'-C3'-O3' torsion angle δ, exhibit an approx. Gaussian distribution centered around the C1'-exo position with δav. = 123° and a range of 79-157°. Purine sugars cluster at high δ values, and pyrimidine sugars cluster at lower δ. A tendency toward 2-fold symmetry in sugar conformation about the center of the mol. is detectable in spite of the destruction of ideal 2-fold symmetry by the mol. bending. More strikingly, sugar conformations of paired bases appear to follow a principle of anticorrelation, with δ values lying approx. the same distance to either side of the center value, δ = 123°. This same anticorrelation is also obsd. in other DNA and DNA·RNA structures.
34德鲁，H. R.， Wing ， RM， 高野， T.， 布罗卡， C.， 田中， S.， 板仓， K.和迪克森，R. E. （1981） B-DNA 十二聚体的结构：构象和动力学 Proc. Natl. Acad. Sci. U. S. A. 78， 2179 DOI： 10.1073/pnas.78.4.2179Google Scholar内容34B-DNA 十二聚体的结构。 I. 体型和动力学Drew， Horace R.;理查德 M.高野常弘;布罗卡，克里斯托弗;田中庄司;板仓，庆一;理查德·迪克森美国国家科学院院刊 （1981 年）、 78 （4）、 2179-83 年科登： 美国国家科学院院刊 （PNASA6）; 国际标准书号：0027-8424。在1.9-Å 分辨率下，将合成 DNA 十二聚体 d（CpGpCpGpApApTpCpGpCpG） 的晶体结构细化为 R = 17.8% 的残差。（2-σ 数据）。 分子略微形成 >1 右旋双链 B 螺旋的完全转数。 螺旋的 2 端与相邻的 mol 重叠并互锁小沟。上下 21 个螺钉轴，在 Dodecimer 的 11 个碱基对步骤上在 Helix 轴上产生 19° 弯曲。 在分子的中心，扰动最小的地方，螺旋线的平均旋转速度为每步 36.9°，或每圈 9.8 个碱基对。 A· 之间的平均螺旋桨扭曲（基面之间的总二面角）分子中心的 T 碱基对为 17.3°，两端 C·G 对之间的碱基对为 avs。11.5°. 通过 C5'-C4'-C3'-O3' 扭转角δ测量的单个脱氧核糖环构象显示大约。高斯分布以 δav 的 C1'-exo 位置为中心。= 123°，范围为 79-157°。 嘌呤糖以高 δ 值聚集，嘧啶糖以较低δ聚集。 尽管摩尔弯曲破坏了理想的 2 倍对称性，但可以检测到糖构象围绕摩尔中心的 2 倍对称性趋势。 更引人注目的是，成对碱基的糖构象似乎遵循反相关原则，δ值与中心值两侧的距离大致相同，δ = 123°。 同样的反相关也是 obsd。在其他 DNA 和 DNA·RNA 结构。 - 35Sponer, J., Jurecka, P., and Hobza, P. (2004) Accurate interaction energies of hydrogen-bonded nucleic acid base pairs J. Am. Chem. Soc. 126, 10142 DOI: 10.1021/ja048436sGoogle Scholar 谷歌学术35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXlvVOltr4%253D&md5=209861454eb2a02c4b70c08abb9abe25Accurate Interaction Energies of Hydrogen-Bonded Nucleic Acid Base PairsSponer, Jiri; Jurecka, Petr; Hobza, PavelJournal of the American Chemical Society (2004), 126 (32), 10142-10151CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Hydrogen-bonded nucleic acids base pairs substantially contribute to the structure and stability of nucleic acids. The study presents ref. ab initio structures and interaction energies of selected base pairs with binding energies ranging from -5 to -47 kcal/mol. The mol. structures are obtained using the RI-MP2 (resoln. of identity MP2) method with extended cc-pVTZ basis set of AOs. The RI-MP2 method provides results essentially identical with the std. MP2 method. The interaction energies are calcd. using the Complete Basis Set (CBS) extrapolation at the RI-MP2 level. For some base pairs, Coupled-Cluster corrections with inclusion of non-iterative triple contributions (CCSD(T)) are given. The calcns. are compared with selected medium quality methods. The PW91 DFT functional with the 6-31G** basis set matches well the RI-MP2/CBS abs. interaction energies and reproduces the relative values of base pairing energies with a max. relative error of 2.6 kcal/mol when applied with Becke3LYP-optimized geometries. The Becke3LYP DFT functional underestimates the interaction energies by few kcal/mol with relative error of 2.2 kcal/mol. Very good performance of nonpolarizable Cornell et al. force field is confirmed and this indirectly supports the view that H-bonded base pairs are primarily stabilized by electrostatic interactions.
35斯波纳，J.， 尤雷卡， P.和 Hobza， P. （2004） 氢键核酸碱基对的精确相互作用能 J. Am. Chem. Soc. 126， 10142 DOI： 10.1021/JA048436sGoogle Scholar内容35氢键核酸碱基对的精确相互作用能Sponer， Jiri;尤雷卡，彼得;Hobza， PavelJournal of the American Chemical Society （美国化学会帕维尔杂志） （2004 年）， 126 元 （32）、 10142-10151科登： JACSAT公司; 国际标准书号：0002-7863。 （美国化学学会）氢键核酸碱基对对核酸的结构和稳定性有很大贡献。 该研究提出了所选碱基对的从头到尾的结构和相互作用能，结合能范围为 -5 至 -47 kcal/mol。 分子结构是使用 RI-MP2 （同一 MP2 解析） 方法和扩展的 cc-pVTZ 基础 AO 集获得的。 RI-MP2 方法提供的结果与标准 MP2 方法基本相同。 相互作用能是 calcd 的。在 RI-MP2 级别使用完整基集 （CBS） 外推。 对于某些碱基对，给出了包含非迭代三重贡献 （CCSD（T）） 的耦合簇校正。 calcns.与选定的中等质量方法进行比较。 具有 6-31G** 基集的 PW91 DFT 与 RI-MP2/CBS 相互作用能很好地匹配，并再现碱基配对能量的相对值，当应用于 Becke3LYP 优化的几何形状时，最大相对误差为 2.6 kcal/mol。 Becke3LYP DFT 泛函低估了几 kcal/mol 的相互作用能，相对误差为 2.2 kcal/mol。 不可极化的 Cornell 等人的力场性能非常好，这间接支持了 H 键合碱对主要通过静电相互作用稳定的观点。 - 36Bak, K. L., Jørgensen, P., Olsen, J., Helgaker, T., and Klopper, W. (2000) Accuracy of atomization energies and reaction enthalpies in standard and extrapolated electronic wave function/basis set calculations J. Chem. Phys. 112, 9229 DOI: 10.1063/1.481544Google Scholar 谷歌学术36https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXjsVSnu7w%253D&md5=0f65812ac2c3cd8da5818cb1500f06f6Accuracy of atomization energies and reaction enthalpies in standard and extrapolated electronic wave function/basis set calculationsBak, Keld L.; Jorgensen, Poul; Olsen, Jeppe; Helgaker, Trygve; Klopper, WimJournal of Chemical Physics (2000), 112 (21), 9229-9242CODEN: JCPSA6; ISSN:0021-9606. (American Institute of Physics)The accuracy of std. ab initio wave function calcns. of atomization energies and reaction enthalpies has been assessed by comparing with exptl. data for 16 small closed-shell mols. and 13 isogyric reactions. The investigated wave function models are Hartree-Fock (HF), Moeller-Plesset second-order perturbation theory (MP2), coupled-cluster theory with singles and doubles excitations (CCSD) and CCSD with perturbative triple-excitation corrections [CCSD(T)]; the one-electron basis sets used are the correlation-consistent cc-pVxZ and cc-pCVxZ basis sets with cardinal nos. x = D, T, Q, 5, and 6. Results close to the basis-set limit have been obtained by using two-point extrapolations. In agreement with previous studies, it is found that the intrinsic error of the CCSD(T) method is less than chem. accuracy (≈4 kJ/mol) for both atomization energies and reaction enthalpies. The mean and max. abs. errors of the best CCSD(T) calcns. are 0.8 and 2.3 kJ/mol for the atomization energies and 1.0 and 2.3 kJ/mol for the reaction enthalpies. Chem. accuracy is obtained already from the extrapolations based on the cc-pCVTZ and cc-pCVQZ basis sets, with mean and max. abs. errors of 1.7 and 4.0 kJ/mol for atomization energies and 1.3 and 3.1 kJ/mol for reaction enthalpies. The intrinsic errors of the Hartree-Fock, MP2, and CCSD wave function models are significantly larger than for CCSD(T). For CCSD and MP2, the mean abs. errors in the basis set limit are about 32 kJ/mol for the atomization energies and about 10 and 15 kJ/mol, resp., for the reaction enthalpies. For the Hartree-Fock model, the mean abs. errors are 405 and 29 kJ/mol for atomization energies and reaction enthalpies, resp. Correlation of the core electrons is important in order to obtain accurate results with CCSD(T). Without compromising the accuracy, the core contribution may be calcd. with a basis set that has one cardinal no. lower than that used for the valence correlation contribution. Basis-set extrapolation should be used for both the core and the valence contributions.
36巴克，KL， Jørgensen， P.， 奥尔森， J.， 赫尔加克， T.和 Klopper， W. （2000） 标准和外推电子波函数/基集计算中原子化能和反应焓的准确性 J. Chem. Phys. 112， 9229 DOI： 10.1063/1.481544Google Scholar36内容？origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXjsVSnu7w%253D&md5=0f65812ac2c3cd8da5818cb1500f06f6标准和外推电子波函数/基集计算中原子化能和反应焓的准确性Bak， Keld L.;乔根森，保罗;奥尔森，杰普;赫尔加克，特里格夫;Klopper， Wim化学物理杂志 （2000 年）、 112 元 （21）、 9229-9242科登： JCPSA6; 国际标准书号：0021-9606。 （美国物理学会）通过与 16 个小闭壳分子和 13 个同轴反应的实验数据进行比较，评估了原子化能和反应焓的标准 ab initio 波函数计算的准确性。 研究的波函数模型是 Hartree-Fock （HF）、Moeller-Plesset 二阶扰动理论 （MP2）、单双激励耦合簇理论 （CCSD） 和扰动三重激励校正 CCSD [CCSD（T）];使用的单电子基集是相关性一致的 cc-pVxZ 和 cc-pCVxZ 基集，基数为 x = D、T、Q、5 和 6。 接近基集极限的结果是通过使用两点外推获得的。 与以前的研究一致，发现 CCSD（T） 方法的内禀误差对于原子化能和反应焓都小于化学精度 （≈4 kJ/mol）。 最佳 CCSD（T） 计算器的平均值和最大绝对误差。原子化能为 0.8 和 2.3 kJ/mol，反应焓为 1.0 和 2.3 kJ/mol。 化学精度已经从基于 cc-pCVTZ 和 cc-pCVQZ 基集的外推中获得，原子化能的平均和最大绝对误差为 1.7 和 4.0 kJ/mol，反应焓为 1.3 和 3.1 kJ/mol。 Hartree-Fock、MP2 和 CCSD 波函数模型的内禀误差明显大于 CCSD（T）。 对于 CCSD 和 MP2，原子化能的基集极限中的平均绝对误差约为 32 kJ/mol，反应焓约为 10 和 15 kJ/mol。 对于 Hartree-Fock 模型，原子化能和反应焓的平均绝对误差分别为 405 和 29 kJ/mol。 核心电子的相关性对于使用 CCSD（T） 获得准确的结果很重要。 在不影响准确性的情况下，核心贡献可以是 calcd。具有一个基集，该基集具有一个基数 NO。低于用于价相关贡献的 URL。 基集外推应用于核心和价贡献。 - 37Watts, J. D., Gauss, J., and Bartlett, R. J. (1993) Coupled-cluster methods with noniterative triple excitations for restricted open-shell Hartree–Fock and other general single determinant reference functions. Energies and analytical gradients J. Chem. Phys. 98, 8718 DOI: 10.1063/1.464480Google Scholar 谷歌学术37https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3sXksFKnsrY%253D&md5=af3b7d1ea8ac357af387d567a5f849c1Coupled-cluster methods with noniterative triple excitations for restricted-open-shell-Hartree-Fock and other general single-determinant reference functions. Energies and analytical gradientsWatts, John D.; Gauss, Juergen; Bartlett, Rodney J.Journal of Chemical Physics (1993), 98 (11), 8718-33CODEN: JCPSA6; ISSN:0021-9606.A new, noniterative triples correction to the CCSD method, for general single determinant ref. functions is proposed and investigated numerically for various cases, including non-Hartree-Fock (non-HF) ref. functions. It is correct through fourth-order of perturbation theory for non-HF refs., and unlike other such methods, retains the usual invariance properties common to CC methods, while requiring only a single N7 step. In the canonical Hartree-Fock case, the method is equiv. to the usual CCSD(T) method, but now permits the use of restricted open-shell Hartree-Fock (ROHF) and quasirestricted Hartree-Fock (QRHF) ref. determinants, along with many others. Comparisons with full CI (FCI) results are presented for CH2, CH2+, CH3, NH2, and SiH2. The derivation and initial computational implementation of anal. gradients for the ROHF-CCSD(T) method, which includes UHF (UHF) CCSD(T) and RHF-CCSD(T) as special cases, are also reported. Applications of anal. gradients are presented for HOO, the CN radical, which is highly spin contaminated at the UHF level, and HCO, the latter with several large basis sets. With these developments of anal. gradients, these highly accurate generalized CCSD(T) methods can be widely applied.
37瓦茨，法学博士， 高斯， J.和巴特利特，RJ。 （1993） 具有非迭代三重激励的耦合集群方法，用于受限开壳 Hartree-Fock 和其他一般的单一行列式参考函数。能量和分析梯度 J. Chem. Phys. 98， 8718 DOI： 10.1063/1.464480Google Scholar内容37https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3sXksFKnsrY%253D&md5=af3b7d1ea8ac357af387d567a5f849c1 的更多具有非迭代三重激励的耦合集群方法，用于受限开壳 Hartree-Fock 和其他一般的单行列式参考函数。能量和分析梯度Watts， John D.;高斯，Juergen;巴特利特，罗德尼 J.化学物理学报 （1993 年）、 98 （11）、 8718-33科登： JCPSA6; 国际标准书号：0021-9606。针对一般的单一行列式参考函数，提出了一种新的非迭代三元组校正 CCSD 方法，并针对各种情况进行了数值研究，包括非 Hartree-Fock （非 HF） 参考函数。 对于非 HF 参考文献，通过扰动理论的四阶是正确的，并且与其他此类方法不同，它保留了 CC 方法常见的通常不变性，同时只需要一个 N7 步骤。 在规范的 Hartree-Fock 案例中，该方法等同于通常的 CCSD（T） 方法，但现在允许使用受限制的开壳 Hartree-Fock （ROHF） 和准受限制的 Hartree-Fock （QRHF） 参考行列式，以及许多其他行列式。 介绍了 CH2 、 CH2 + 、 CH3 、 NH2 和 SiH2 与全 CI （FCI） 结果的比较。 还报道了 ROHF-CCSD（T） 方法的分析梯度的推导和初始计算实现，其中包括 UHF （UHF） CCSD（T） 和 RHF-CCSD（T） 作为特殊情况。 分析梯度的应用介绍了 HOO（CN 自由基，在 UHF 水平上受到高度自旋污染）和 HCO（后者具有几个大碱基集）。 随着分析梯度的这些发展，这些高度准确的广义 CCSD（T） 方法可以得到广泛应用。 - 38Coester, F. and Kümmel, H. (1960) Short-Range Correlations in Nuclear Wave Functions Nucl. Phys. 17, 477 DOI: 10.1016/0029-5582(60)90140-1Google Scholar 谷歌学术38https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaF3MXmvFSlsw%253D%253D&md5=e99794469699b0b7595035ce600d6476Short-range correlations in nuclear wave functionsCoester, F.; Kummel, H.Nuclear Physics (1960), 17 (), 477-85CODEN: NUPHA7; ISSN:0029-5582.The 1- and 2-body d. matrixes are well approximated by expressions involving only the single-particle wave functions and the 2-body cluster functions. The Schr.ovrddot.odinger equation yielded a coupled set of equations which detd. the cluster functions as well as the single-particle wave functions.
38科斯特，F. 和 Kümmel， H. （1960） 核波函数中的短程相关性 Nucl. Phys. 17， 477 DOI： 10.1016/0029-5582（60）90140-1Google Scholar内容38https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaF3MXmvFSlsw%253D%253D&md5=e99794469699b0b7595035ce600d6476 的更多核波函数中的短程相关性Coester， F.;库梅尔，H.核物理 》（1960 年）、 17 （）， 477-85 元科登： 努帕7; 国际标准书号：0029-5582。1 体和 2 体 d. 矩阵通过仅涉及单粒子波函数和 2 体集群函数的表达式很好地近似。 Schr.ovrddot.odinger 方程产生了一组耦合方程，其中 detd.Cluster 函数以及 Single-particle Wave 函数。 - 39Purvis, G. D. and Bartlett, R. J. (1982) A full coupled-cluster singles and doubles model: The inclusion of disconnected triples J. Chem. Phys. 76, 1910 DOI: 10.1063/1.443164Google Scholar 谷歌学术39https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL38XhtFSgtLY%253D&md5=0cf9405a65eed10485f6fc00646ef037A full coupled-cluster singles and doubles model: the inclusion of disconnected triplesPurvis, George D., III; Bartlett, Rodney J.Journal of Chemical Physics (1982), 76 (4), 1910-18CODEN: JCPSA6; ISSN:0021-9606.The coupled-cluster singles and doubles model (CCSD) is derived algebraically, presenting the full set of equations for a general ref. function explicitly in spin-orbital form. The computational implementation of the CCSD model, which involves cubic and quartic terms, is discussed and results are reported and compared with full CI calcns. for H2O and BeH2. The CCSD exponential ansatz sums higher-order correlation effects efficiently even for BeH2, near its transition state geometry where quasidegeneracy efforts are quite large, recovering 98% of the full CI correlation energy. For H2O, CCSD plus the 4th-order triple excitation correction agrees with the full CI energy to 0.5 kcal/mol. Comparisons with low-order models provide ests. of the effect of the higher-order terms on the correlation energy.
39珀维斯，GD 和巴特利特，RJ。 （1982） 一个完整的耦合集群单双模型：包含断开连接的三元组 J. Chem. Phys. 76， 1910 DOI： 10.1063/1.443164Google Scholar内容39https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL38XhtFSgtLY%253D&md5=0cf9405a65eed10485f6fc00646ef037 的更多全耦合集群单双模型：包含不相连的三元组Purvis， George D.， III;巴特利特，罗德尼 J.化学物理学报 （1982 年）、 76 （4）、 1910 年 18 月科登： JCPSA6; 国际标准书号：0021-9606。耦合簇单双模型 （CCSD） 是通过代数推导的，以自旋轨道形式明确表示一般参考函数的全套方程。 讨论了涉及三次项和四次项的 CCSD 模型的计算实现，并报告了结果并与完整的 CI 计算进行了比较。用于 H 2 O 和 BeH 2。 CCSD 指数拟设有效地求和了高阶相关效应，即使对于 BeH2，接近其过渡态几何，其中 quasidegeneracy 努力相当大，恢复了 98% 的完整 CI 相关能量。 对于 H 2 O，CCSD 加上 4 阶三重激发校正与完整的 CI 能量一致，达到 0.5 kcal/mol。 与低阶模型的比较提供了 ests。高阶项对相关能的影响。 - 40Jorgensen, W. L. and Pranata, J. (1990) Importance of secondary interactions in triply hydrogen bonded complexes: guanine-cytosine vs uracil-2,6-diaminopyridine J. Am. Chem. Soc. 112, 2008 DOI: 10.1021/ja00161a061Google Scholar 谷歌学术40https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3cXhtFymsLc%253D&md5=2a10cf61aecf583270135a87f7c5adcbImportance of secondary interactions in triply hydrogen bonded complexes: guanine-cytosine vs uracil-2,6-diaminopyridineJorgensen, William L.; Pranata, JuliantoJournal of the American Chemical Society (1990), 112 (5), 2008-10CODEN: JACSAT; ISSN:0002-7863.Although complexes of guanine-cytosine and uracil-2,6-diaminopyridine are both triply H-bonded in CHCl3, measured assocn. consts. for such systems vary by 102-103. The origin of the discrepancy is analyzed here through computational studies. Monte Carlo statistical mechanics simulations for the complexes in CHCl3 also find the substantial binding preference for guanine-cytosine. The difference is then traced to the gas-phase interaction energies which favor guanine-cytosine complexation by ca. 10 kcal/mol. The three H bonds are of the same type in both complexes; however, the variation in their arrangement leads to secondary electrostatic effects that account for the destabilization of the uracil-2,6-diaminopyridine complex. Such secondary interactions are a significant element for consideration in mol. design.
40乔根森，WL 和 Pranata， J. （1990） 三氢键配合物中次级相互作用的重要性：鸟嘌呤-胞嘧啶与尿嘧啶-2,6-二氨基吡啶 J. Am. Chem. Soc. 112， 2008 DOI： 10.1021/JA00161A061Google Scholar内容40https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3cXhtFymsLc%253D&md5=2a10cf61aecf583270135a87f7c5adcb 的更多三氢键配合物中次级相互作用的重要性：鸟嘌呤-胞嘧啶与尿嘧啶-2,6-二氨基吡啶Jorgensen， William L.;Pranata， JuliantoJournal of the American Chemical Society （美国化学会杂志） （1990 年）、 112 元 （5）、 2008 年 10 月科登： JACSAT公司; 国际标准书号：0002-7863。尽管鸟嘌呤-胞嘧啶和尿嘧啶-2,6-二氨基吡啶的复合物在 CHCl3 中都是三重 H 键合的，但此类系统的测量 assocn. const. 差异 102-103。 这里通过计算研究分析了这种差异的根源。 CHCl3 中复合物的蒙特卡罗统计力学模拟也发现鸟嘌呤-胞嘧啶的结合偏好很大。 然后将差异追溯到气相反作用能，该能有利于鸟嘌呤-胞嘧啶络合约 10 kcal/mol。 三个 H 键在两个复合物中属于同一类型;然而，它们排列的变化会导致次级静电效应，从而导致尿嘧啶-2,6-二氨基吡啶复合物不稳定。 这种次级相互作用是分子设计中需要考虑的重要因素。 - 41Wang, W., Sheng, X., Zhang, S., Huang, F., Sun, C., Liu, J., and Chen, D. (2016) Theoretical characterization of the conformational features of unnatural oligonucleotides containing a six nucleotide genetic alphabet Phys. Chem. Chem. Phys. 18, 28492 DOI: 10.1039/C6CP05594J
41王 W.， 盛 X.， 张 S.， 黄 F.， 太阳， C.， 刘 J.和 Chen， D. （2016） 包含六核苷酸遗传字母的非天然寡核苷酸构象特征的理论表征 Phys. Chem. Chem. Phys. 18， 28492 DOI： 10.1039/C6CP05594J - 42Wang, X., Hoshika, S., Peterson, R. J., Kim, M. J., Benner, S. A., and Kahn, J. D. (2017) Biophysics of Artificially Expanded Genetic Information Systems. Thermodynamics of DNA Duplexes Containing Matches and Mismatches Involving 2-Amino-3-nitropyridin-6-one (Z) and Imidazo[1,2-a]-1,3,5-triazin-4(8H)one (P) ACS Synth. Biol. 6, 782 DOI: 10.1021/acssynbio.6b00224Google Scholar 谷歌学术42https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtVKhtbg%253D&md5=8621b81e603d75d3079a67bf3768ae1bBiophysics of Artificially Expanded Genetic Information Systems. Thermodynamics of DNA Duplexes Containing Matches and Mismatches Involving 2-Amino-3-nitropyridin-6-one (Z) and Imidazo[1,2-a]-1,3,5-triazin-4(8H)one (P)Wang, Xiaoyu; Hoshika, Shuichi; Peterson, Raymond J.; Kim, Myong-Jung; Benner, Steven A.; Kahn, Jason D.ACS Synthetic Biology (2017), 6 (5), 782-792CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)Synthetic nucleobases presenting non-Watson-Crick arrangements of hydrogen bond donor and acceptor groups can form addnl. nucleotide pairs that stabilize duplex DNA independent of the std. A:T and G:C pairs. The pair between 2-amino-3-nitropyridin-6-one 2'-deoxyriboside (presenting a {donor-donor-acceptor} hydrogen bonding pattern on the Watson-Crick face of the small component, trivially designated Z) and imidazo[1,2-a]-1,3,5-triazin-4(8H)one 2'-deoxyriboside (presenting an {acceptor-acceptor-donor} hydrogen bonding pattern on the large component, trivially designated P) is one of these extra pairs for which a substantial amt. of mol. biol. has been developed. Here, we report the results of UV absorbance melting measurements and det. the energetics of binding of DNA strands contg. Z and P to give short duplexes contg. Z:P pairs as well as various mismatches comprising Z and P. All measurements were done at 1 M NaCl in buffer (10 mM Na cacodylate, 0.5 mM EDTA, pH 7.0). Thermodn. parameters (ΔH°, ΔS°, and ΔG°37) for oligonucleotide hybridization were extd. Consistent with the Watson-Crick model that considers both geometric and hydrogen bonding complementarity, the Z:P pair was found to contribute more to duplex stability than any mismatches involving either nonstandard nucleotide. Further, the Z:P pair is more stable than a C:G pair. The Z:G pair was found to be the most stable mismatch, forming either a deprotonated mismatched pair or a wobble base pair analogous to the stable T:G mismatch. The C:P pair is less stable, perhaps analogous to the wobble pair obsd. for C:O6-methyl-G, in which the pyrimidine is displaced into the minor groove. The Z:A and T:P mismatches are much less stable. Parameters for predicting the thermodn. of oligonucleotides contg. Z and P bases are provided. This represents the first case where this has been done for a synthetic genetic system.
42王 X.， 星香， S.， 彼得森， RJ， 金， MJ， 本纳， SA和 Kahn， J. D. （2017） 人工扩展遗传信息系统的生物物理学。含有涉及 2-氨基-3-硝基吡啶-6-酮 （Z） 和咪唑[1,2-A]-1,3,5-三嗪-4（8H）酮 （P） 的匹配和错配的 DNA 双链体的热力学 ACS Synth. Biol. 6， 782 DOI： 10.1021/acssynbio.6b00224Google Scholar42内容？origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtVKhtbg%253D&md5=8621b81e603d75d3079a67bf3768ae1b人工扩展遗传信息系统的生物物理学。含有 2-氨基-3-硝基吡啶-6-酮 （Z） 和咪唑[1,2-a]-1,3,5-三嗪-4（8H）酮 （P） 的 DNA 双链体的热力学 （P）Wang， Xiaoyu;星香，Shuichi;彼得森，雷蒙德 J.;Kim， Myong-Jung;本纳，史蒂文 A.;卡恩，杰森 D.ACS 合成生物学 （2017 年）， 6 （5）、 782-792科登： ASBCD6; 国际标准书号：2161-5063。 （美国化学学会）呈现氢键供体和受体基团的非 Watson-Crick 排列的合成核碱基可以形成额外的核苷酸对，从而独立于标准因素稳定双链 DNA。A：T 和 G：C 对。 2-氨基-3-硝基吡啶-6-酮 2'-脱氧核苷（在小组分的 Watson-Crick 面上呈现 {供体-供体-受体} 氢键模式，通常命名为 Z）和咪唑[1,2-a]-1,3,5-三嗪-4（8H）一 2'-脱氧核糖苷（在大组分上呈现 {受体-受体-供体}氢键模式，通常命名为 P）之间的对是这些额外的对之一，已经开发了大量的分子生物学。 在这里，我们报告了紫外吸光度熔解测量的结果，并描述了 DNA 链结合的能量学 contg。Z 和 P 给出短双工 contg。Z：P 对以及构成 Z 和 P 的各种不匹配。 所有测量均在 1 M NaCl 缓冲液（10 mM 二甲胂酸钠，0.5 mM EDTA，pH 7.0）中进行。 Thermodn.寡核苷酸杂交的参数 （ΔH°、ΔS° 和 ΔG°37） 已 EXTD 。 与考虑几何键和氢键互补性的 Watson-Crick 模型一致，发现 Z：P 对对双链体稳定性的贡献大于涉及任一非标准核苷酸的任何错配。 此外，Z：P 对比 C：G 对更稳定。 发现 Z：G 对是最稳定的错配，形成去质子化错配对或类似于稳定的 T：G 错配的摆动碱基对。 C：P 对不太稳定，可能类似于摆动对 obsd。对于 C：O 6-甲基-G，其中嘧啶被置换到小沟中。 Z：A 和 T：P 失配的稳定性要差得多。 用于预测 thermodn 的参数。 寡核苷酸 contg。提供 Z 和 P 底座。 这是对合成遗传系统进行此操作的第一个案例。 - 43Malyshev, D. A., Pfaff, D. A., Ippoliti, S. I., Hwang, G. T., Dwyer, T. J., and Romesberg, F. E. (2010) Solution structure, mechanism of replication, and optimization of an unnatural base pair Chem. - Eur. J. 16, 12650 DOI: 10.1002/chem.201000959Google Scholar 谷歌学术43https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhtlCksrnI&md5=da5cf1e3c584834fe385ea153c05f7bcSolution Structure, Mechanism of Replication, and Optimization of an Unnatural Base PairMalyshev, Denis A.; Pfaff, Danielle A.; Ippoliti, Shannon I.; Hwang, Gil Tae; Dwyer, Tammy J.; Romesberg, Floyd E.Chemistry - A European Journal (2010), 16 (42), 12650-12659, S12650/1-S12650/28CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)As part of an ongoing effort to expand the genetic alphabet for in vitro and eventual in vivo applications, we have synthesized a wide variety of predominantly hydrophobic unnatural base pairs and evaluated their replication in DNA. Collectively, the results have led us to propose that these base pairs, which lack stabilizing edge-on interactions, are replicated by means of a unique intercalative mechanism. Here, we report the synthesis and characterization of three novel derivs. of the nucleotide analog dMMO2, which forms an unnatural base pair with the nucleotide analog d5SICS. Replacing the para-Me substituent of dMMO2 with an annulated furan ring (yielding dFMO) has a dramatically neg. effect on replication, while replacing it with a methoxy (dDMO) or with a thiomethyl group (dTMO) improves replication in both steady-state assays and during PCR amplification. Thus, dTMO-d5SICS, and esp. dDMO-d5SICS, represent significant progress toward the expansion of the genetic alphabet. To elucidate the structure-activity relationships governing unnatural base pair replication, we detd. the soln. structure of duplex DNA contg. the parental dMMO2-d5SICS pair, and also used this structure to generate models of the deriv. base pairs. The results strongly support the intercalative mechanism of replication, reveal a surprisingly high level of specificity that may be achieved by optimizing packing interactions, and should prove invaluable for the further optimization of the unnatural base pair.
43马雷舍夫，D. A.， 普法夫， DA， 伊波利蒂， S. I.， 黄， GT， 德怀尔， TJ和 Romesberg， F. E. （2010） 非天然碱基对的溶液结构、复制机制和优化 Chem. - Eur. J. 16， 12650 DOI： 10.1002/化学201000959Google Scholar43内容 https://chemport.cas.org/services/resolver？origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhtlCksrnI&md5=da5cf1e3c584834fe385ea153c05f7bc非自然碱基对的溶液结构、复制机制和优化Malyshev， Denis A.;普法夫，丹妮尔 A.;Ippoliti， 香农 I.;黄吉泰;德怀尔，塔米 J.;罗梅斯伯格，弗洛伊德 E.化学 - A European Journal （2010 年）， 16 （42）、 12650-12659、S12650/1-S12650/28科登： CEUJED; 国际标准书号：0947-6539。 （Wiley-VCH Verlag GmbH & Co. KGaA）作为扩展体外和最终体内应用的遗传字母表的持续努力的一部分，我们合成了多种主要疏水性的非天然碱基对，并评估了它们在 DNA 中的复制。 总的来说，结果使我们提出，这些缺乏稳定边缘相互作用的碱基对是通过独特的插层机制来复制的。 在这里，我们报告了三个新衍生物的综合和表征。核苷酸类似物 dMMO2 的 dMMO2 与核苷酸类似物 d5SICS 形成非天然碱基对。 用环状呋喃环取代 dMMO2 的对位 Me 取代基（产生 dFMO）对复制有显著的负效应，而用甲氧基 （dDMO） 或硫代甲基 （dTMO） 取代它可改善稳态检测和 PCR 扩增过程中的复制。 因此，dTMO-d5SICS 和 dDMO-d5SICS 代表了遗传字母表扩展的重大进展。 为了阐明控制非自然碱基对复制的结构-活性关系，我们 detd.双链体 DNA contg 的结构。亲本 dMMO2-d5SICS 对，并且还使用此结构生成 Deriv. 碱基对的模型。 结果强烈支持复制的插层机制，揭示了通过优化包装相互作用可以实现的令人惊讶的高特异性水平，并且应该证明对于进一步优化非天然碱基对非常有价值。 - 44Kimoto, M., Kawai, R., Mitsui, T., Yokoyama, S., and Hirao, I. (2009) An unnatural base pair system for efficient PCR amplification and functionalization of DNA molecules Nucleic Acids Res. 37, e14 DOI: 10.1093/nar/gkn956Google Scholar 谷歌学术44https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtlGksL8%253D&md5=8812c6b48c1a2c611efdd4f77afda742An unnatural base pair system for efficient PCR amplification and functionalization of DNA moleculesKimoto, Michiko; Kawai, Rie; Mitsui, Tsuneo; Yokoyama, Shigeyuki; Hirao, IchiroNucleic Acids Research (2009), 37 (2), e14/1-e14/9CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)Toward the expansion of the genetic alphabet, we present an unnatural base pair system for efficient PCR amplification, enabling the site-specific incorporation of extra functional components into DNA. This system can be applied to conventional PCR protocols employing DNA templates contg. unnatural bases, natural and unnatural base triphosphates, and a 3'→5' exonuclease-proficient DNA polymerase. For highly faithful and efficient PCR amplification involving the unnatural base pairing, we identified the natural-base sequences surrounding the unnatural bases in DNA templates by an in vitro selection technique, using a DNA library contg. the unnatural base. The system facilitates the site-specific incorporation of a variety of modified unnatural bases, linked with functional groups of interest, into amplified DNA. DNA fragments (0.15 amol) contg. the unnatural base pair can be amplified 107-fold by 30 cycles of PCR, with <1% total mutation rate of the unnatural base pair site. Using the system, we demonstrated efficient PCR amplification and functionalization of DNA fragments for the extremely sensitive detection of zeptomol-scale target DNA mols. from mixts. with excess amts. (pmol scale) of foreign DNA species. This unnatural base pair system will be applicable to a wide range of DNA/RNA-based technologies.
44木本 M.， 河合， R.， 三井， T.， 横山， S.和 Hirao， I. （2009） 用于 DNA 分子高效 PCR 扩增和功能化的非天然碱基对系统核酸研究 37， e14 DOI： 10.1093/NAR/GKN956Google Scholar内容44一种用于 DNA 分子高效 PCR 扩增和功能化的非天然碱基对系统Kimoto， Michiko;河合，理惠;三井津雄;横山茂之;Hirao， Ichiro核酸研究 （2009 年）、 37 元 （2）， 编号 E14/1-E14/9科登： 纳尔哈德; 国际标准书号：0305-1048。 （牛津大学出版社）为了扩展遗传字母表，我们提出了一种用于高效 PCR 扩增的非天然碱基对系统，能够将额外的功能成分特异性掺入 DNA 中。 该系统可应用于采用 DNA 模板的常规 PCR 方案。非天然碱基、天然和非天然碱基三磷酸盐以及 3'→5' 核酸外切酶熟练的 DNA 聚合酶。 为了涉及非自然碱基配对的高度忠实和高效的 PCR 扩增，我们使用 DNA 文库 contg 通过体外选择技术鉴定了 DNA 模板中非天然碱基周围的天然碱基序列。不自然的基底。 该系统有助于将与目标官能团相连的各种修饰的非天然碱基进行位点特异性掺入扩增的 DNA 中。 DNA 片段 （0.15 amol） 连续。非自然碱基对可通过 30 次 PCR 循环扩增 107 倍，非自然碱基对位点的总突变率为 <1%。 使用该系统，我们展示了 DNA 片段的高效 PCR 扩增和功能化，用于对 zeptomol 级靶 DNA mols 的极其灵敏的检测。来自 Mixts。外源 DNA 物种的 AMTS. （PMOL scale）。 这种非天然碱基对系统将适用于广泛的基于 DNA/RNA 的技术。 - 45Yamashige, R., Kimoto, M., Takezawa, Y., Sato, A., Mitsui, T., Yokoyama, S., and Hirao, I. (2012) Highly specific unnatural base pair systems as a third base pair for PCR amplification Nucleic Acids Res. 40, 2793 DOI: 10.1093/nar/gkr1068Google Scholar 谷歌学术45https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XkvFOku70%253D&md5=cb29171678a521654ea4966f14365018Highly specific unnatural base pair systems as a third base pair for PCR amplificationYamashige, Rie; Kimoto, Michiko; Takezawa, Yusuke; Sato, Akira; Mitsui, Tsuneo; Yokoyama, Shigeyuki; Hirao, IchiroNucleic Acids Research (2012), 40 (6), 2793-2806CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)Toward the expansion of the genetic alphabet of DNA, we present highly efficient unnatural base pair systems as an artificial third base pair for PCR. Hydrophobic unnatural base pair systems between 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) and 2-nitro-4-propynylpyrrole (Px) were fine-tuned for efficient PCR, by assessing the amplification efficiency and fidelity using different polymerases and template sequence contexts and modified Px bases. Then, we found that some modifications of the Px base reduced the misincorporation rate of the unnatural base substrates opposite the natural bases in templates without reducing the Ds-Px pairing selectivity. Under optimized conditions using Deep Vent DNA polymerase, the misincorporation rate was extremely low (0.005%/bp/replication), which is close to that of the natural base mispairings by the polymerase. DNA fragments with different sequence contexts were amplified ∼1010-fold by 40 cycles of PCR, and the selectivity of the Ds-Px pairing was >99.9%/replication, except for 99.77%/replication for unfavorable purine-Ds-purine motifs. Furthermore, >97% of the Ds-Px pair in DNA survived in the 1028-fold amplified products after 100-cycle PCR (10 cycles repeated 10 times). This highly specific Ds-Px pair system provides a framework for new biotechnol.
45山重 R.， 木本， M.， 竹泽， Y.， 佐藤， A.， 三井， T.， 横山， S.和 Hirao， I. （2012） 高度特异性的非天然碱基对系统作为 PCR 扩增的第三个碱基对 Nucleic Acids Res. 40， 2793 DOI： 10.1093/NAR/GKR1068Google Scholar内容45https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XkvFOku70%253D&md5=cb29171678a521654ea4966f14365018 的更多高度特异性的非天然碱基对系统作为 PCR 扩增的第三个碱基对Yamashige， Rie;木本美智子;竹泽雄介;佐藤，阿基拉;三井津雄;横山茂之;Hirao， Ichiro核酸研究 （2012 年）、 40 （6）、 2793-2806科登： 纳尔哈德; 国际标准书号：0305-1048。 （牛津大学出版社）为了扩展 DNA 的遗传字母表，我们提出了高效的非天然碱基对系统作为 PCR 的人工第三碱基对。 通过使用不同的聚合酶和模板序列上下文以及修饰的 Px 碱基来评估扩增效率和保真度，对 7-（2-噻吩基）咪唑并[4,5-b]吡啶 （Ds） 和 2-硝基-4-丙炔基吡咯 （Px） 之间的疏水性非天然碱基对系统进行了微调，以实现高效 PCR。 然后，我们发现 Px 碱基的一些修饰降低了模板中与天然碱基相反的非天然碱基底物的错误掺入率，而没有降低 Ds-Px 配对选择性。 在使用 Deep Vent DNA 聚合酶的优化条件下，错误掺入率极低 （0.005%/bp/复制），接近聚合酶的天然碱基错配率。 通过 40 个循环的 PCR 将具有不同序列背景的 DNA 片段扩增 ∼1010 倍，Ds-Px 配对的选择性为 >99.9%/复制，除了不利的嘌呤-Ds-嘌呤基序的 99.77%/复制。 此外，DNA 中 Ds-Px 对的 >97% 在 100 个循环 PCR 后在 1028 倍扩增产物中存活（10 个循环重复 10 次）。 这种高度特异性的 Ds-Px 对系统为新的生物技术提供了一个框架。 - 46Betz, K., Kimoto, M., Diederichs, K., Hirao, I., and Marx, A. (2017) Structural Basis for Expansion of the Genetic Alphabet with an Artificial Nucleobase Pair Angew. Chem., Int. Ed. DOI: 10.1002/anie.201706478
46贝茨，K.， 木本， M.， 迪德里希斯， K.， 平尾， I.和 Marx， A. （2017） 用人工核碱基对扩展遗传字母表的结构基础 Angew. Chem.， Int. Ed. DOI： 10.1002/anie.201706478 - 47Kirnos, M. D., Khudyakov, I. Y., Alexandrushkina, N. I., and Vanyushin, B. F. (1977) 2-aminoadenine is an adenine substituting for a base in S-2L cyanophage DNA Nature 270, 369 DOI: 10.1038/270369a0Google Scholar 谷歌学术47https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE1cXhtVWhtbk%253D&md5=cda382dcaf4600a055736d1c2daf6d252-Aminoadenine is an adenine substituting for a base in S-2L cyanophage DNAKirnos, M. D.; Khudyakov, I. Y.; Alexandrushkina, N. I.; Vanyushin, B. F.Nature (London, United Kingdom) (1977), 270 (5635), 369-70CODEN: NATUAS; ISSN:0028-0836.Full enzymic hydrolysis of DNA from cyanophage S-2L revealed that deoxyadenine was completely substituted for by 2-aminoadenine deoxyribonucleotide (I). The DNA contained I, deoxythymidine, deoxycytidine, and deoxyguanidine in molar percentages 15.9, 15.4, 34.4, and 3.43, resp. The addnl. amino group offered by I permits the formation of 3 H-bonds in the I-thymidine base pair; this accounts for the higher Tm of 85.6° for the I-contg. S-2L DNA compared with 82.0° for adenine-contg. DNA.
47基尔诺斯，医学博士， 胡迪亚科夫， I. Y.， 亚历山德鲁什金娜， N. I.和 Vanyushin， BF。 （1977） 2-氨基腺嘌呤是腺嘌呤替代 S-2L 蓝噬细胞 DNA 中的碱基 Nature 270， 369 DOI： 10.1038/270369A0Google Scholar内容47https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE1cXhtVWhtbk%253D&md5=cda382dcaf4600a055736d1c2daf6d25 的更多2-氨基腺嘌呤是 S-2L 蓝噬细胞 DNA 中碱基的腺嘌呤替代Kirnos， M. D.;Khudyakov， I. Y.;亚历山德鲁什金娜，N. I.;Vanyushin， BFNature （英国 伦敦） （1977 年）、 270 （5635）、 369-70 元科登： 纳图阿斯; 国际标准书号：0028-0836。来自蓝噬菌体 S-2L 的 DNA 的完全酶解显示，脱氧腺嘌呤完全被 2-氨基腺嘌呤脱氧核糖核苷酸 （I） 取代。 DNA 含有 I、脱氧胸苷、脱氧胞苷和脱氧胍，摩尔百分比为 15.9、15.4、34.4 和 3.43。 I 提供的附加氨基允许在 I-胸苷碱基对中形成 3 个 H 键;这解释了 I-contg 的 Tm 高达 85.6°。S-2L DNA 与腺嘌呤结合 82.0° 的比较。脱氧核糖核酸。 - 48Howard, F. B. and Miles, H. T. (1984) 2NH2A X T helices in the ribo- and deoxypolynucleotide series. Structural and energetic consequences of 2NH2A substitution Biochemistry 23, 6723 DOI: 10.1021/bi00321a068
48霍华德，FB 和 H. T. 的迈尔斯。 （1984） 核糖多核苷酸和脱氧多核苷酸系列中的 2NH2A X T 螺旋。2NH2A 取代的结构和能量后果 生物化学 23， 6723 DOI： 10.1021/BI00321A068 - 49Minor, W., Cymborowski, M., Otwinowski, Z., and Chruszcz, M. (2006) HKL-3000: the integration of data reduction and structure solution--from diffraction images to an initial model in minutes Acta Crystallogr., Sect. D: Biol. Crystallogr. 62, 859 DOI: 10.1107/S0907444906019949Google Scholar 谷歌学术49https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XntVWksL8%253D&md5=2cd2fe288f954837abb4acae4c008a2fHKL-3000: the integration of data reduction and structure solution - from diffraction images to an initial model in minutesMinor, Wladek; Cymborowski, Marcin; Otwinowski, Zbyszek; Chruszcz, MaksymilianActa Crystallographica, Section D: Biological Crystallography (2006), D62 (8), 859-866CODEN: ABCRE6; ISSN:0907-4449. (Blackwell Publishing Ltd.)A new approach that integrates data collection, data redn., phasing and model building significantly accelerates the process of structure detn. and on av. minimizes the no. of data sets and synchrotron time required for structure soln. Initial testing of the HKL-3000 system (the beta version was named HKL-2000_ph) with more than 140 novel structure detns. has proven its high value for MAD/SAD expts. The heuristics for choosing the best computational strategy at different data resoln. limits of phasing signal and crystal diffraction are being optimized. The typical end result is an interpretable electron-d. map with a partially built structure and, in some cases, an almost complete refined model. The current development is oriented towards very fast structure soln. in order to provide feedback during the diffraction expt. Work is also proceeding towards improving the quality of phasing calcn. and model building.
49米诺，W.， 辛博罗夫斯基， M.， 奥特温诺夫斯基， Z.和 Chruszcz， M. （2006） HKL-3000：数据缩减和结构解决方案的集成--在几分钟内从衍射图像到初始模型Acta Crystallogr.， Sect. D： Biol. Crystallogr. 62， 859 DOI： 10.1107/S0907444906019949Google Scholar内容49https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XntVWksL8%253D&md5=2cd2fe288f954837abb4acae4c008a2f 的更多HKL-3000： 数据缩减和结构解决方案的集成 - 在几分钟内从衍射图像到初始模型Minor， Wladek;辛博罗夫斯基，马尔辛;Otwinowski， Zbyszek;Chruszcz，MaksymilianActa Crystallographica，D 部分：生物晶体学 （2006 年）、 D62 系列 （8）、 859-866科登： ABCRE6; 国际标准书号：0907-4449。 （布莱克威尔出版有限公司）一种集成数据收集、数据重置、定相和模型构建的新方法大大加快了结构定义和 AV 的过程，最大限度地减少了结构构建所需的数据集数量和同步加速器时间。 HKL-3000 系统（测试版命名为 HKL-2000_ph）的初步测试，有 140 多个新结构设计。已经证明了它对 MAD/SAD 测试的高价值。 在不同数据分辨率下选择最佳计算策略的启发式方法。相位信号和晶体衍射的极限正在优化中。 典型的最终结果是可解释的 electron-d。具有部分构建结构的地图，在某些情况下，具有几乎完整的精致模型。 目前的开发方向是非常快速的结构解决方案。以便在衍射实验期间提供反馈。 提高 Phasing calcn 的质量的工作也在进行中。和模型构建。 - 50Vagin, A. and Teplyakov, A. (2010) Molecular replacement with MOLREP Acta Crystallogr., Sect. D: Biol. Crystallogr. 66, 22 DOI: 10.1107/S0907444909042589Google Scholar 谷歌学术50https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXit1Kktw%253D%253D&md5=820d114719aca209994ffb0403e3b20dMolecular replacement with MOLREPVagin, Alexei; Teplyakov, AlexeiActa Crystallographica, Section D: Biological Crystallography (2010), 66 (1), 22-25CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)MOLREP is an automated program for mol. replacement that utilizes a no. of original approaches to rotational and translational search and data prepn. Since the first publication describing the program, MOLREP has acquired a variety of features that include weighting of the X-ray data and search models, multi-copy search, fitting the model into electron d., structural superposition of two models and rigid-body refinement. The program can run in a fully automatic mode using optimized parameters calcd. from the input data.
50阴道，A. 和 Teplyakov， A. （2010） 用 MOLREP Acta Crystallogr.， Sect. D： Biol. Crystallogr. 66， 22 DOI： 10.1107/S0907444909042589Google Scholar内容50https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXit1Kktw%253D%253D&md5=820d114719aca209994ffb0403e3b20d 的更多用 MOLREP 阴道替代分子，Alexei;Teplyakov， AlexeiActa Crystallographica， Section D： 生物晶体学 （2010 年）， 66 （1）、 22-25 元科登： ABCRE6; 国际标准书号：0907-4449。 （国际晶体学联合会）MOLREP 是一个用于 mol. replacement 的自动化程序，它利用大量原始方法进行旋转和平移搜索以及数据准备。 自首次发表描述该程序以来，MOLREP 已经获得了各种功能，包括 X 射线数据和搜索模型的加权、多副本搜索、将模型拟合到电子 d.、两个模型的结构叠加和刚体细化。 该程序可以使用优化参数 calcd 以全自动模式运行。从输入数据中。 - 51Emsley, P., Lohkamp, B., Scott, W. G., and Cowtan, K. (2010) Features and development of Coot Acta Crystallogr., Sect. D: Biol. Crystallogr. 66, 486 DOI: 10.1107/S0907444910007493Google Scholar 谷歌学术51https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXksFKisb8%253D&md5=67262cbfc60004de5ef962d5c043c910Features and development of CootEmsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K.Acta Crystallographica, Section D: Biological Crystallography (2010), 66 (4), 486-501CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)Coot is a mol.-graphics application for model building and validation of biol. macromols. The program displays electron-d. maps and at. models and allows model manipulations such as idealization, real-space refinement, manual rotation/translation, rigid-body fitting, ligand search, solvation, mutations, rotamers and Ramachandran idealization. Furthermore, tools are provided for model validation as well as interfaces to external programs for refinement, validation and graphics. The software is designed to be easy to learn for novice users, which is achieved by ensuring that tools for common tasks are 'discoverable' through familiar user-interface elements (menus and toolbars) or by intuitive behavior (mouse controls). Recent developments have focused on providing tools for expert users, with customisable key bindings, extensions and an extensive scripting interface. The software is under rapid development, but has already achieved very widespread use within the crystallog. community. The current state of the software is presented, with a description of the facilities available and of some of the underlying methods employed.
51埃姆斯利，P.， 洛坎普， B.， 斯科特， WG和 Cowtan， K. （2010） Coot Acta Crystallogr. 的特征和发展，D 节：Biol. Crystallogr. 66， 486 DOI： 10.1107/S0907444910007493Google Scholar内容51CootEmsley， P. 的特点和发展;洛坎普，B.;斯科特，WG;考坦，K.Acta Crystallographica，D 部分：生物晶体学 （2010 年）， 66 （4）、 邮编 486-501科登： ABCRE6; 国际标准书号：0907-4449。 （国际晶体学联合会）Coot 是一种分子图形应用程序，用于生物大分子的模型构建和验证。 程序将显示 electron-d。maps 和 at.模型并允许模型操作，例如理想化、实空间细化、手动旋转/平移、刚体拟合、配体搜索、溶剂化、突变、旋转体和拉氏理想化。 此外，还提供了用于模型验证的工具以及用于优化、验证和图形的外部程序接口。 该软件旨在让新手用户易于学习，这是通过确保通过熟悉的用户界面元素（菜单和工具栏）或直观的行为（鼠标控制）“可发现”常见任务的工具来实现的。 最近的开发集中在为专家用户提供工具，具有可自定义的键绑定、扩展和广泛的脚本界面。 该软件正在快速开发中，但已经在 crystallog 中实现了非常广泛的使用。社区。 提供了软件的当前状态，并描述了可用的设施和所采用的一些基础方法。 - 52Murshudov, G. N., Vagin, A. A., and Dodson, E. J. (1997) Refinement of macromolecular structures by the maximum-likelihood method Acta Crystallogr., Sect. D: Biol. Crystallogr. 53, 240 DOI: 10.1107/S0907444996012255Google Scholar 谷歌学术52https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXjs1Gnsb4%253D&md5=ec7f141ce1542f7ff458b98ecfe3f8afRefinement of macromolecular structures by the maximum-likelihood methodMurshudov, Garib N.; Vagin, Alexei A.; Dodson, Eleanor J.Acta Crystallographica, Section D: Biological Crystallography (1997), D53 (3), 240-255CODEN: ABCRE6; ISSN:0907-4449. (Munksgaard)A review with many refs. on the math. basis of max. likelihood. The likelihood function for macromol. structures is extended to include prior phase information and exptl. std. uncertainties. The assumption that different parts of a structure might have different errors is considered. A method for estg. σA using "free" reflections is described and its effects analyzed. The derived equations have been implemented in the program REFMAC. This has been tested on several proteins at different stages of refinement (bacterial α-amylase, cytochrome c', cross-linked insulin and oligopeptide binding protein). The results derived using the max.-likelihood residual are consistently better than those obtained from least-squares refinement.
52穆尔舒多夫，G. N.， 阴道， AA和 Dodson， EJ （1997） 通过最大似然法细化大分子结构学报，D部分：生物学晶体学家。53， 240 DOI： 10.1107/S0907444996012255Google Scholar的更多内容52https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXjs1Gnsb4%253D&md5=ec7f141ce1542f7ff458b98ecfe3f8af通过最大似然法细化大分子结构Murshudov， Garib N.;阴道，阿列克谢 A.;多德森，埃莉诺 J.Acta Crystallographica，D 部分：生物晶体学 （1997 年）、 D53 系列 （3）、 240-255 元科登： ABCRE6; 国际标准书号：0907-4449。 （蒙克斯加德）一篇基于最大似然度的数学基础的评论，有许多参考文献。 macromol 的似然函数。结构扩展为包括先前阶段信息和 exptl. std. 不确定性。 考虑了结构的不同部分可能具有不同误差的假设。 estg 的方法。描述了使用“自由”反射的 σA 并分析了其影响。 推导的方程已在 REFMAC 程序中实现。 这已经在不同精炼阶段的几种蛋白质（细菌 α-淀粉酶、细胞色素 c'、交联胰岛素和寡肽结合蛋白）上进行了测试。 使用最大似然残差得出的结果始终优于从最小二乘细化中获得的结果。 - 53Adams, P. D., Afonine, P. V., Bunkoczi, G., Chen, V. B., Davis, I. W., Echols, N., Headd, J. J., Hung, L. W., Kapral, G. J., Grosse-Kunstleve, R. W., McCoy, A. J., Moriarty, N. W., Oeffner, R., Read, R. J., Richardson, D. C., Richardson, J. S., Terwilliger, T. C., and Zwart, P. H. (2010) PHENIX: a comprehensive Python-based system for macromolecular structure solution Acta Crystallogr., Sect. D: Biol. Crystallogr. 66, 213 DOI: 10.1107/S0907444909052925Google Scholar 谷歌学术53https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhs1Sisbc%253D&md5=67b439ff4bd61c659cae37ca4209b7bcPHENIX: a comprehensive Python-based system for macromolecular structure solutionAdams, Paul D.; Afonine, Pavel V.; Bunkoczi, Gabor; Chen, Vincent B.; Davis, Ian W.; Echols, Nathaniel; Headd, Jeffrey J.; Hung, Li Wei; Kapral, Gary J.; Grosse-Kunstleve, Ralf W.; McCoy, Airlie J.; Moriarty, Nigel W.; Oeffner, Robert; Read, Randy J.; Richardson, David C.; Richardson, Jane S.; Terwilliger, Thomas C.; Zwart, Peter H.Acta Crystallographica, Section D: Biological Crystallography (2010), 66 (2), 213-221CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)A review. Macromol. X-ray crystallog. is routinely applied to understand biol. processes at a mol. level. However, significant time and effort are still required to solve and complete many of these structures because of the need for manual interpretation of complex numerical data using many software packages and the repeated use of interactive three-dimensional graphics. PHENIX has been developed to provide a comprehensive system for macromol. crystallog. structure soln. with an emphasis on the automation of all procedures. This has relied on the development of algorithms that minimize or eliminate subjective input, the development of algorithms that automate procedures that are traditionally performed by hand and, finally, the development of a framework that allows a tight integration between the algorithms.
53亚当斯，P. D.， 阿福宁， PV， Bunkoczi， G.， 陈， V. B.， 戴维斯， IW， 埃科尔斯， N.， Headd， J. J.， 洪 L. W.， 卡普拉尔， GJ， Grosse-Kunstleve， RW， 麦考伊， AJ， 莫里亚蒂， NW， 奥夫纳， R.， 里德， RJ， 理查森， D. C.， 理查森， JS， 特威利格， TC和 Zwart， PH （2010） PHENIX：用于大分子结构解决方案的基于 Python 的综合系统Acta Crystallogr.， Sect. D： Biol. Crystallogr. 66， 213 DOI： 10.1107/S0907444909052925Google Scholar内容53PHENIX：一个基于 Python 的综合系统，用于大分子结构解决方案Adams， Paul D.;Afonine， Pavel V.;Bunkoczi， Gabor;陈文森特 B.;戴维斯，伊恩 W.;埃科尔斯，纳撒尼尔;负责人 Jeffrey J.;洪丽伟;卡普拉尔，加里 J.;Grosse-Kunstleve， Ralf W.;麦考伊，艾尔利 J.;莫里亚蒂，奈杰尔 W.;奥夫纳，罗伯特;里德，兰迪 J.;理查森，大卫 C.;理查森，简 S.;特威利格，托马斯 C.;彼得 H. 兹瓦特Acta Crystallographica，D 部分：生物晶体学 （2010 年）， 66 （2）， 页码： 213-221法典： ABCRE6; 国际标准书号：0907-4449。 （国际晶体学联合会）评论。 大分子。X 射线晶体学。通常用于了解 mol.此时，S 然而，由于需要使用许多软件包手动解释复杂的数值数据，并且重复使用交互式三维图形，因此仍然需要大量的时间和精力来解决和完成其中的许多结构。 PHENIX 的开发旨在为 macromol 提供全面的系统。crystallog 的另一方面，治安官办公室强调所有程序的自动化。 这依赖于最小化或消除主观输入的算法的开发，自动化传统上手动执行的程序的算法的开发，最后，允许算法之间紧密集成的框架的开发。 - 54Zhao, Y. and Truhlar, D. G. (2008) The M06 suite of density functionals for main group thermochemistry, thermochemical kinetics, noncovalent interactions, excited states, and transition elements: two new functionals and systematic testing of four M06-class functionals and 12 other functionals Theor. Chem. Acc. 120, 215 DOI: 10.1007/s00214-007-0310-xGoogle Scholar 谷歌学术54https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXltFyltbY%253D&md5=c31d6f319d7c7a45aa9b716220e4a422The M06 suite of density functionals for main group thermochemistry, thermochemical kinetics, noncovalent interactions, excited states, and transition elements: two new functionals and systematic testing of four M06-class functionals and 12 other functionalsZhao, Yan; Truhlar, Donald G.Theoretical Chemistry Accounts (2008), 120 (1-3), 215-241CODEN: TCACFW; ISSN:1432-881X. (Springer GmbH)We present two new hybrid meta exchange-correlation functionals, called M06 and M06-2X. The M06 functional is parametrized including both transition metals and nonmetals, whereas the M06-2X functional is a high-nonlocality functional with double the amt. of nonlocal exchange (2X), and it is parametrized only for nonmetals. The functionals, along with the previously published M06-L local functional and the M06-HF full-Hartree-Fock functionals, constitute the M06 suite of complementary functionals. We assess these four functionals by comparing their performance to that of 12 other functionals and Hartree-Fock theory for 403 energetic data in 29 diverse databases, including ten databases for thermochem., four databases for kinetics, eight databases for noncovalent interactions, three databases for transition metal bonding, one database for metal atom excitation energies, and three databases for mol. excitation energies. We also illustrate the performance of these 17 methods for three databases contg. 40 bond lengths and for databases contg. 38 vibrational frequencies and 15 vibrational zero point energies. We recommend the M06-2X functional for applications involving main-group thermochem., kinetics, noncovalent interactions, and electronic excitation energies to valence and Rydberg states. We recommend the M06 functional for application in organometallic and inorganometallic chem. and for noncovalent interactions.
54赵 Y. 和 Truhlar， D. G. （2008） 用于主族热化学、热化学动力学、非共价相互作用、激发态和过渡元素的 M06 密度泛函套件：两个新的泛函和四个 M06 类泛函和 12 个其他泛函的系统测试。化学 Acc. 120， 215 DOI： 10.1007/s00214-007-0310-xGoogle Scholar内容54https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXltFyltbY%253D&md5=c31d6f319d7c7a45aa9b716220e4a422 的更多用于主族热化学、热化学动力学、非共价相互作用、激发态和过渡元素的 M06 密度泛函套件：两个新的泛函和 4 个 M06 类官能团和 12 个其他官能团的系统测试Zhao， Yan;特鲁拉尔，唐纳德 G.理论化学账户 （2008 年）、 120 元 （1-3）、 215-241 元科登： TCACFW; 国际标准书号：1432-881X。 （施普林格有限公司）我们提出了两个新的混合元交换相关函数，称为 M06 和 M06-2X。 M06 泛函参数化包括过渡金属和非金属，而 M06-2X 泛函是高度非局域性泛函，其量是非局域交换量 （2X） 的两倍，并且仅针对非金属参数化。 这些泛函与之前发布的 M06-L 局部泛函和 M06-HF 全 Hartree-Fock 泛函一起构成了 M06 互补泛函套件。 我们通过将这四种泛函的性能与其他 12 个泛函和 Hartree-Fock 理论的性能进行比较来评估 29 个不同数据库中的 403 个能量数据，包括 10 个热化学数据库、4 个动力学数据库、8 个非共价相互作用数据库、3 个过渡金属键数据库、1 个金属原子激发能数据库和 3 个摩尔激发能数据库。 我们还说明了这 17 种方法对三个数据库的性能 contg。40 个键长和数据库 contg.38 个振动频率和 15 个振动零点能量。 我们推荐将 M06-2X 官能团用于涉及主族热化学、动力学、非共价相互作用以及价态和里德堡态的电子激发能的应用。 我们推荐将 M06 官能团用于有机金属和无机金属化学以及非共价相互作用。 - 55Kendall, R. A., Dunning, T. H., Jr, and Harrison, R. J. (1992) Electron affinities of the first-row atoms revisited. Systematic basis sets and wave functions J. Chem. Phys. 96, 6796 DOI: 10.1063/1.462569Google Scholar 谷歌学术55https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK38XktFClurw%253D&md5=948a06eee10604a8fa37eae2b2ada4beElectron affinities of the first-row atoms revisited. Systematic basis sets and wave functionsKendall, Rick A.; Dunning, Thom H., Jr.; Harrison, Robert J.Journal of Chemical Physics (1992), 96 (9), 6796-806CODEN: JCPSA6; ISSN:0021-9606.The authors describe a reliable procedure for calcg. the electron affinity of an atom and present results for H, B, C, O, and F (H is included for completeness). This procedure involves the use of the recently proposed correlation-consistent basis sets augmented with functions to describe the more diffuse character of the at. anion coupled with a straightforward, uniform expansion of the ref. space for multireference singles and doubles configuration-interaction (MRSD-CI) calcns. A comparison is given with previous results and with corresponding full CI calcns. The most accurate EAs obtained from the MRSD-CI calcns. are (with exptl. values in parentheses): H 0.740 eV (0.754), B 0.258 (0.277), C 1.245 (1.263), O 1.384 (1.461), and F 3.337 (3.401). The EAs obtained from the MR-SDCI calcns. differ by less than 0.03 eV from those predicted by the full CI calcns.
55肯德尔，RA、Dunning， TH， Jr 和 Harrison， RJ （1992） 重新审视第一行原子的电子亲和力。系统基集和波函数 J. Chem. Phys. 96， 6796 DOI： 10.1063/1.462569Google Scholar的更多内容55重新审视第一行原子的电子亲和力。 系统基集和波函数Kendall， Rick A.;邓宁，小汤姆 H.;哈里森，罗伯特 J.化学物理学报 （1992 年）、 96 （9）、 6796-806科登： JCPSA6; 国际标准书号：0021-9606。作者描述了一种可靠的计算方法。原子的电子亲和力，并给出了 H、B、C、O 和 F 的结果（为了完整起见，包括 H）。 此过程涉及使用最近提出的相关性一致基集，并通过函数进行增强，以描述 at 的更分散的特征。阴离子与多参考单双配型相互作用 （MRSD-CI） 计算的参考空间的直接、均匀扩展相结合。 与以前的结果和相应的完整 CI 计算进行了比较。 从 MRSD-CI 计算中获得的最准确的 EA。是（括号中带有说明值）：H 0.740 eV （0.754）、B 0.258 （0.277）、C 1.245 （1.263）、O 1.384 （1.461） 和 F 3.337 （3.401）。 从 MR-SDCI 计算器获得的 EA。与完整 CI 计算预测的差异小于 0.03 eV。 - 56Frisch, M. J., Trucks, G. W., Schlegel, H. B., Scuseria, G. E., Robb, M. A., Cheeseman, J. R., Scalmani, G., Barone, V., Mennucci, B., Petersson, G. A., Nakatsuji, H., Caricato, M., Li, X., Hratchian, H. P., Izmaylov, A. F., Bloino, J., Zheng, G., Sonnenberg, J. L., Hada, M., Ehara, M., Toyota, K., Fukuda, R., Hasegawa, J., Ishida, M., Nakajima, T., Honda, Y., Kitao, O., Nakai, H., Vreven, T., Montgomery, J. A., Jr., Peralta, J. E., Ogliaro, F., Bearpark, M., Heyd, J. J., Brothers, E., Kudin, K. N., Staroverov, V. N., Kobayashi, R., Normand, J., Raghavachari, K., Rendell, A., Burant, J. C., Iyengar, S. S., Tomasi, J., Cossi, M., Rega, N., Millam, J. M., Klene, M., Knox, J. E., Cross, J. B., Bakken, V., Adamo, C., Jaramillo, J., Gomperts, R., Stratmann, R. E., Yazyev, O., Austin, A. J., Cammi, R., Pomelli, C., Ochterski, J. W., Martin, R. L., Morokuma, K., Zakrzewski, V. G., Voth, G. A., Salvador, P., Dannenberg, J. J., Dapprich, S., Daniels, A. D., Farkas, O., Foresman, J. B., Ortiz, J. V., Cioslowski, J., and Fox, D. J. (2009) Gaussian 09, revision E.01, Gaussian, Inc., Wallingford, CT.Google Scholar 谷歌学术There is no corresponding record for this reference.
56Frisch， MJ， 卡车， GW， Schlegel， HB， Scuseria， GE， Robb， MA， Cheeseman， JR， Scalmani， G.， Barone， V.， Mennucci， B.， Petersson， GA， Nakatsuji， H.， Caricato， M.， Li， X.， Hratchian， HP， Izmaylov， AF， Bloino， J.， Zheng， G.， Sonnenberg， JL， Hada， M.， Ehara， M.， 丰田， K.， Fukuda， R.， 长谷川， J.， 石田，M.，中岛，T.，本田，Y.，北尾，O.，中井，H.，Vreven，T.，蒙哥马利，JA，Jr.，佩拉尔塔，JE，奥利亚罗，F.，Bearpark，M.，Heyd，JJ，兄弟，E.，Kudin，KN，Staroverov，VN，小林，R.，诺曼德，J.，Raghavachari，K.，伦德尔，A.，布兰特，JC，艾扬格，SS，托马西，J.，科西，M.，雷加，N.，米勒姆，JM， 克莱恩，M.，诺克斯，JE，克罗斯，JB，巴肯，V.，阿达莫，C.，贾拉米洛，J.，贡佩茨，R.，斯特拉特曼，RE，亚兹耶夫，O.，奥斯汀，AJ，卡米，R.，波梅利，C.，奥赫特斯基，JW，马丁，RL，Morokuma，K.，Zakrzewski，VG，沃斯，GA，萨尔瓦多，P.，丹能伯格，JJ，达普里奇，S.，丹尼尔斯，AD，法卡斯，O.，福斯曼，JB， Ortiz, J. V., Cioslowski, J., and Fox, D. J. (2009) Gaussian 09, revision E.01, Gaussian, Inc., Wallingford, CT. - 59Řezáč, J. and Hobza, P. (2016) Benchmark Calculations of Interaction Energies in Noncovalent Complexes and Their Applications Chem. Rev. 116, 5038 DOI: 10.1021/acs.chemrev.5b00526Google Scholar 谷歌学术59https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xjs1yqsb4%253D&md5=09e9bdc03605dac8bc2054321c71480dBenchmark Calculations of Interaction Energies in Noncovalent Complexes and Their ApplicationsRezac, Jan; Hobza, PavelChemical Reviews (Washington, DC, United States) (2016), 116 (9), 5038-5071CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. Data sets of benchmark interaction energies in noncovalent complexes are an important tool for quantifying the accuracy of computational methods used in this field, as well as for the development of new computational approaches. This review is intended as a guide to conscious use of these data sets. We discuss their construction and accuracy, list the data sets available in the literature, and demonstrate their application to validation and parametrization of quantum-mech. computational methods. In practical model systems, the benchmark interaction energies are usually obtained using composite CCSD(T)/CBS schemes. To use these results as a benchmark, their accuracy should be estd. first. We analyze the errors of this methodol. with respect to both the approxns. involved and the basis set size. We list the most prominent data sets covering various aspects of the field, from general ones to sets focusing on specific types of interactions or systems. The benchmark data are then used to validate more efficient computational approaches, including those based on explicitly correlated methods. Special attention is paid to the transition to large systems, where accurate benchmarking is difficult or impossible, and to the importance of nonequil. geometries in parametrization of more approx. methods.
59热扎奇 J. 和 Hobza， P. （2016） 非共价配合物中相互作用能的基准计算及其应用 Chem. Rev. 116， 5038 DOI： 10.1021/acs.chemrev.5b00526Google Scholar内容59非共价配合物中相互作用能的基准计算及其应用Rezac， Jan;Hobza， PavelChemical Reviews （美国 华盛顿特区） （2016 年）、 116 元 （9）、 邮编：5038-5071科登： 克雷; 国际标准书号：0009-2665。 （美国化学学会）评论。 非共价配合物中基准相互作用能的数据集是量化该领域所用计算方法的准确性以及开发新计算方法的重要工具。 本综述旨在为有意识地使用这些数据集提供指南。 我们讨论了它们的构造和准确性，列出了文献中可用的数据集，并演示了它们在量子机械验证和参数化中的应用。计算方法。 在实际模型系统中，基准相互作用能通常是使用复合 CCSD（T）/CBS 方案获得的。 要将这些结果用作基准，应首先估计其准确性。 我们分析了这种方法的误差。相对于这两个近似值。涉及和基集大小。 我们列出了涵盖该领域各个方面的最突出的数据集，从一般数据集到专注于特定类型的交互或系统的数据集。 然后，基准数据用于验证更高效的计算方法，包括基于显式相关方法的计算方法。 特别注意向大型系统的过渡，其中很难或不可能进行准确的基准测试，以及 nonequil 的重要性。参数化的几何图形 More Approx. 方法。 - 60Lotrich, V. F., Flocke, N., Ponton, M., Yau, A. D., Perera, A. S., Deumens, E., and Bartlett, R. J. (2008) Parallel implementation of electronic structure energy, gradient, and Hessian calculations J. Chem. Phys. 128, 194104 DOI: 10.1063/1.2920482Google Scholar 谷歌学术60https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXmt12qtL0%253D&md5=007f341da25be49d1aed06ecf0db15e4Parallel implementation of electronic structure energy, gradient, and Hessian calculationsLotrich, V.; Flocke, N.; Ponton, M.; Yau, A. D.; Perera, A.; Deumens, E.; Bartlett, R. J.Journal of Chemical Physics (2008), 128 (19), 194104/1-194104/15CODEN: JCPSA6; ISSN:0021-9606. (American Institute of Physics)ACES III is a newly written program in which the computationally demanding components of the computational chem. code ACES II; [ACES II program system, University of Florida, 1994] have been redesigned and implemented in parallel. The high-level algorithms include Hartree-Fock (HF) SCF, second-order many-body perturbation theory [MBPT(2)] energy, gradient, and Hessian, and coupled cluster singles, doubles, and perturbative triples [CCSD(T)] energy and gradient. For SCF, MBPT(2), and CCSD(T), both restricted HF and unrestricted HF ref. wave functions are available. For MBPT(2) gradients and Hessians, a restricted open-shell HF ref. is also supported. The methods are programmed in a special language designed for the parallelization project. The language is called super instruction assembly language (SIAL). The design uses an extreme form of object-oriented programing. All compute intensive operations, such as tensor contractions and diagonalizations, all communication operations, and all input-output operations are handled by a parallel program written in C and FORTRAN 77. This parallel program, called the super instruction processor (SIP), interprets and executes the SIAL program. By sepg. the algorithmic complexity (in SIAL) from the complexities of execution on computer hardware (in SIP), a software system is created that allows for very effective optimization and tuning on different hardware architectures with quite manageable effort. (c) 2008 American Institute of Physics.
60洛特里希，V. F.， 弗洛克， N.， 庞顿， M.， 丘， A. D.， 佩雷拉， A. S.， 杜门斯， E.和巴特利特，RJ。 （2008） 电子结构能量、梯度和 Hessian 计算的并行实现，J. Chem. Phys. 128， 194104 DOI： 10.1063/1.2920482Google Scholar60内容？origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXmt12qtL0%253D&md5=007f341da25be49d1aed06ecf0db15e4电子结构能量、梯度和 Hessian 计算的并行实现Lotrich， V.;弗洛克，N.;庞顿，M.;丘桐，A. D.;佩雷拉，A.;Deumens， E.;巴特利特，RJ化学物理学报 （2008 年）、 票价 128 元 （19）、 194104/1-194104/15科登： JCPSA6; 国际标准书号：0021-9606。 （美国物理学会）ACES III 是一个新编写的程序，其中计算化学的计算要求高的组件代码 ACES II;[ACES II 程序系统，佛罗里达大学，1994 年]已经重新设计并并行实施。 高级算法包括 Hartree-Fock （HF） SCF、二阶多体微扰理论 [MBPT（2）] 能量、梯度和 Hessian 函数，以及耦合簇单、双精度和扰动三元 [CCSD（T）] 能量和梯度。 对于 SCF、MBPT（2） 和 CCSD（T），可以使用受限 HF 和非受限 HF 参考波功能。 对于 MBPT（2） 梯度和 Hessians，还支持受限制的开壳 HF 参考。 这些方法使用专为并行化项目设计的特殊语言进行编程。 该语言称为超级指令汇编语言 （SIAL）。 该设计使用了一种极端形式的面向对象编程。 所有计算密集型操作（例如张量收缩和对角化）、所有通信操作以及所有输入输出操作都由用 C 和 FORTRAN 77 编写的并行程序处理。 这个并行程序称为超级指令处理器 （SIP），用于解释和执行 SIAL 程序。 由 sepg.算法复杂性（在 SIAL 中）从计算机硬件（在 SIP 中）执行的复杂性中，创建了一个软件系统，该系统允许在不同的硬件架构上以相当可管理的努力进行非常有效的优化和调整。 （c） 2008 年美国物理学会。
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Abstract 抽象
Figure 1 图 1
Figure 1. Chemical structures with atom numbers for A:T, G:C, P:Z, and X:P.
Figure 2
Figure 2. Characterization of duplex DNA including X:K pairs by circular dichroism (CD). The ellipticity is plotted versus the wavelength for 2X2K (long dash line), 3X3K (dash dot dot line) along with control sequences for GC (dotted line), and AT (solid line). All of the duplexes have CD spectra indicative of right-handed B-form DNA.
Figure 3
Figure 3. Crystal structure of host–guest complex including self-complementary 16 base pair oligonucleotide. (A) The N-terminal fragment of Moloney murine leukemia virus reverse transcriptase serves as the host in the complex including two protein molecules, shown as cartoon renderings in cyan and green, and a 16-mer duplex, each strand shown as a stick rendering C, cyan or green, O, red, N, blue, and phosphorus in orange; X:K pairs are shown with C in magenta. The complex depicted is that of the host–guest complex for the oligonucleotide shown with two consecutive X:K nucleobase pairs (2X2K). Within our crystals, the asymmetric unit includes only half of the complex depicted and thus the equivalent of eight nucleobase pairs and one protein molecule, indicated by the dashed line. (B) The sequence of the 2X2K sequence and position numbering for the nucleobases within the duplex. (C) The final 2Fo–Fc electron density map is shown as gray mesh renderings contoured at 1.0 σ for the X:K pair at position 6.
Figure 4
Figure 4. van der Waals sphere renderings are shown with O in red, N, blue, P, orange, and C in green for X:K, yellow for P:Z, cyan for G:C, and pink for A:T for major and minor groove presentation faces of the nucleobase pairs.
Figure 5
Figure 5. Comparison of helical properties for host–guest complexes including X:K, G:C, A:T, and P:Z. The unique 8-mer DNA structure including X:K pairs (green) is shown superimposed in (A) with A:T (gray), (B) with G:C (blue), and (C) with P:Z (yellow). (D) The associated minor groove widths for the 16-mer DNA structures are shown in the same colors as designated in stuctures A–C.
Figure 6
Figure 6. Comparison of local base pair properties of X:K and A:T. (A) Cartoon/stick renderings for X:K and A:T pairs (7th nucleotide step, X7:K10 as shown in Figure 3) with a semitransparent gray molecular surface superimposed show significant propeller twist angles for the base plane of X or A vs K or T. A similar rendering is shown for a relatively planar G:C base pair (6th nucleotide step G6:C11). (B) Stacking of XX/KK and AA/TT shown as stick renderings are overall similar in accommodating the nucleobase pairs with significant propeller twist angles.
Figure 7
Figure 7. Dipole moments and electrostatic potential maps (ESPs) for (A) X:K, (B) A:T, (C) G:C, (D) P:Z nucleobase pairs. Methyl substituents are used in place of the C1′ carbon in the deoxyribose ring. The ESP color gradation is such that red is ca. −40 kcal/mol and blue is ca. +40 kcal/mol; strict energetic interpretation is limited to indications of broad differences in reactivity. Dipole moments are shown as a blue arrow, following the convention that a positive vector points toward a positive charge density. The magnitude of the vectors is not proportional to length (length modified for ease of view).
Figure 8
Figure 8. Calculated X:K pair has a proton transfer. (A) The chemical structure is shown for the calculated X:K pair with bond distances in the vicinity of the hydrogen bonding pairs. Bond lengths (Å) of key species for Watson–Crick binding of X:K. All calculations are based on M06-2X/aug″-cc-pVDZ geometries, expected to be accurate to within 0.03 Å. Note the proton transfer from X to K, as shown by the bond lengths. (B) The calculated electron density is shown for the X:K pair and clearly shows that the proton involved in the central hydrogen bonding pair is associated with N3 of K and not N1 of X. Electron density contours given at 0.04 electron charge/Bohr3 from the KS-DFT density matrix.
References
This article references 60 other publications.
- 1Ghosh, A. and Bansal, M. (2003) A glossary of DNA structures from A to Z Acta Crystallogr., Sect. D: Biol. Crystallogr. 59, 620 DOI: 10.1107/S09074449030032511https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXit12kt7Y%253D&md5=9d1306e34c1930e36d870b596f28ee55A glossary of DNA structures from A to ZGhosh, Anirban; Bansal, ManjuActa Crystallographica, Section D: Biological Crystallography (2003), D59 (4), 620-626CODEN: ABCRE6; ISSN:0907-4449. (Blackwell Munksgaard)A review. The right-handed double-helical Watson-Crick model for B-form DNA is the most commonly known DNA structure. In addn. to this classic structure, several other forms of DNA have been obsd., and it is clear that the DNA mol. can assume different structures depending on the base sequence and environment. The various forms of DNA have been identified as A, B, C etc. In fact, a detailed inspection of the literature reveals that only the letters F, Q, U, V and Y are now available to describe any new DNA structure that may appear in the future. It is also apparent that it may be more relevant to talk about the A, B or C type dinucleotide steps, since several recent structures show mixts. of various different geometries and a careful anal. is essential before identifying it as a 'new structure'. This review provides a glossary of currently identified DNA structures and is quite timely as it outlines the present understanding of DNA structure exactly 50 yr after the original discovery of DNA structure by Watson and Crick.
- 2Switzer, C., Moroney, S. E., and Benner, S. A. (1989) Enzymatic incorporation of a new base pair into DNA and RNA J. Am. Chem. Soc. 111, 8322 DOI: 10.1021/ja00203a0672https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1MXls12mtr8%253D&md5=53ace25f0d8cc4aa3a6be39a90a328c0Enzymatic incorporation of a new base pair into DNA and RNASwitzer, Christopher; Moroney, Simon E.; Benner, Steven A.Journal of the American Chemical Society (1989), 111 (21), 8322-3CODEN: JACSAT; ISSN:0002-7863.The Klenow fragment of DNA polymerase I (Escherichia coli) and phage T7 RNA polymerase were found to direct the incorporation of isoguanosine (iso-G) into an oligonucleotide opposite isocytidine (iso-C). Further, expts. were carried out with the Klenow enzyme to det. the specificity with which the new bases pair. On the basis of these expts., it was detd. that essentially no deoxyguanosine or deoxyadenosine was incorporated opposite d-iso-C, and that whereas d-iso-G showed undesired pairing with deoxycytidine. Due to the specificity obsd. in the enzymic incorporation of d-iso-G into DNA, it was concluded that these 2 mols. form a base-pair with a H-bonding pattern distinct from those occurring in the natural A-T(U) and G-C pairs.
- 3Piccirilli, J. A., Krauch, T., Moroney, S. E., and Benner, S. A. (1990) Enzymatic incorporation of a new base pair into DNA and RNA extends the genetic alphabet Nature 343, 33 DOI: 10.1038/343033a03https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3cXitFamu7k%253D&md5=1abf50139303a7ffa31262cdeb736fe5Enzymic incorporation of a new base pair into DNA and RNA extends the genetic alphabetPiccirilli, Joseph A.; Krauch, Tilman; Moroney, Simon E.; Benner, Steven A.Nature (London, United Kingdom) (1990), 343 (6253), 33-7CODEN: NATUAS; ISSN:0028-0836.A new Watson-Crick base pair [κ-xanthine or 7-Me oxyformycin B, where κ = 3-β-D-ribofuranosyl-(2,6-diaminopyrimidine) with a H bonding pattern different from that in the A·T and G·C base pairs, is incorporated into duplex DNA and RNA by DNA and RNA polymerases and expands the genetic alphabet from 4 to 6 letters. This expansion could lead to RNAs with greater diversity in functional groups and greater catalytic potential.
- 4Benner, S. A. (2004) Understanding nucleic acids using synthetic chemistry Acc. Chem. Res. 37, 784 DOI: 10.1021/ar040004z4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXntVOrsb8%253D&md5=ce508328dbf0543bdd7f3b79bda79b84Understanding Nucleic Acids Using Synthetic ChemistryBenner, Steven A.Accounts of Chemical Research (2004), 37 (10), 784-797CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)A review describing work done in these labs. that has used synthetic, phys. org., and biol. chem. to understand the roles played by the nucleobases, sugars, and phosphates of DNA in the mol. recognition processes central to genetics. The no. of nucleobases has been increased from 4 to 12, generating an artificially expanded genetic information system. This system is used today in the clinic to monitor the levels of HIV and hepatitis C viruses in patients, helping to manage patient care. Work with uncharged phosphate replacements suggests that a repeating charge is a universal feature of genetic mols. operating in water and will be found in extraterrestrial life (if it is ever encountered). The use of ribose may reflect prebiotic processes in the presence of borate-contg. minerals, which stabilize ribose formed from simple org. precursors. A new field, synthetic biol., is emerging on the basis of these expts., where chem. mimics biol. processes as complicated as Darwinian evolution.
- 5Geyer, C. R., Battersby, T. R., and Benner, S. A. (2003) Nucleobase pairing in expanded Watson-Crick-like genetic information systems Structure 11, 1485 DOI: 10.1016/j.str.2003.11.0085https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXps1OrtL0%253D&md5=4fdcf5a3d34c73b118c90f57246cbc11Nucleobase Pairing in Expanded Watson-Crick-like Genetic Information SystemsGeyer, C. Ronald; Battersby, Thomas R.; Benner, Steven A.Structure (Cambridge, MA, United States) (2003), 11 (12), 1485-1498CODEN: STRUE6; ISSN:0969-2126. (Cell Press)To guide the design of alternative genetic systems, we measured melting temps. of DNA duplexes contg. matched and mismatched nucleobase pairs from natural and unnatural structures. The pairs were analyzed in terms of structural features, including nucleobase size, no. of hydrogen bonds formed, the presence of uncompensated hydrogen bonding functional groups, the nature of the bond joining the nucleobase to the sugar, and nucleobase charge. The results suggest that stability of nucleobase pairs correlates with the no. of H-bonds, size complementarity, the presence of uncompensated functional groups, and the presence of charge on a nucleobase. Each of these properties appear to be more significant than the nature of the glycosidic bond and sequence context. The results provide guidelines for constructing stable Watson-Crick like nucleobase pairs with unnatural nucleobases. The expts. also demonstrate that expanded genetic systems can be constructed using size complementary nucleobase pairs that contain three hydrogen bonds.
- 6Yakovchuk, P., Protozanova, E., and Frank-Kamenetskii, M. D. (2006) Base-stacking and base-pairing contributions into thermal stability of the DNA double helix Nucleic Acids Res. 34, 564 DOI: 10.1093/nar/gkj4546https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XhtlOnuro%253D&md5=2c72728f600373744c60a81684a64409Base-stacking and base-pairing contributions into thermal stability of the DNA double helixYakovchuk, Peter; Protozanova, Ekaterina; Frank-Kamenetskii, Maxim D.Nucleic Acids Research (2006), 34 (2), 564-574CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)Two factors are mainly responsible for the stability of the DNA double helix: base pairing between complementary strands and stacking between adjacent bases. By studying DNA mols. with solitary nicks and gaps we measure temp. and salt dependence of the stacking free energy of the DNA double helix. For the first time, DNA stacking parameters are obtained directly (without extrapolation) for temps. from below room temp. to close to melting temp. We also obtain DNA stacking parameters for different salt concns. ranging from 15 to 100 mM Na+. From stacking parameters of individual contacts, we calc. base-stacking contribution to the stability of A•T- and G•C-contg. DNA polymers. We find that temp. and salt dependences of the stacking term fully det. the temp. and the salt dependence of DNA stability parameters. For all temps. and salt concns. employed in present study, base-stacking is the main stabilizing factor in the DNA double helix. A•T pairing is always destabilizing and G•C pairing contributes almost no stabilization. Base-stacking interaction dominates not only in the duplex overall stability but also significantly contributes into the dependence of the duplex stability on its sequence.
- 7Benner, S. A., Karalkar, N. B., Hoshika, S., Laos, R., Shaw, R. W., Matsuura, M., Fajardo, D., and Moussatche, P. (2016) Alternative Watson-Crick Synthetic Genetic Systems Cold Spring Harbor Perspect. Biol. 8, a023770 DOI: 10.1101/cshperspect.a0237707https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXpslWktrY%253D&md5=db3376ae594ae9894e2901926bf65133Alternative Watson-Crick synthetic genetic systemsBenner, Steven A.; Karalkar, Nilesh B.; Hoshika, Shuichi; Laos, Roberto; Shaw, Ryan W.; Matsuura, Mariko; Fajardo, Diego; Moussatche, PatriciaCold Spring Harbor Perspectives in Biology (2016), 8 (11), a023770/1-a023770/27CODEN: CSHPEU; ISSN:1943-0264. (Cold Spring Harbor Laboratory Press)In its "grand challenge" format in chem., "synthesis" as an activity sets out a goal that is substantially beyond current theor. and technol. capabilities. In pursuit of this goal, scientists are forced across uncharted territory, where they must answer unscripted questions and solve unscripted problems, creating new theories and new technologies in ways that would not be created by hypothesis-directed research. Thus, synthesis drives discoveryand paradigm changes in waysthat anal. cannot. Described here are the products that have arisen so far through the pursuit of one grand challenge in synthetic biol.: Recreate the genetics, catalysis, evolution, and adaptation that we value in life, but using genetic and catalytic biopolymers different from those that have been delivered to us by natural historyon Earth. The outcomes in technol. include new diagnostic toolsthat have helped personalize the care of hundreds of thousands of patients worldwide. In science, the effort has generated a fundamentally different view of DNA, RNA, and how they work.
- 8Lutz, M. J., Horlacher, J., and Benner, S. A. (1998) Recognition of a non-standard base pair by thermostable DNA polymerases Bioorg. Med. Chem. Lett. 8, 1149 DOI: 10.1016/S0960-894X(98)00177-2There is no corresponding record for this reference.
- 9Yang, Z., Chen, F., Chamberlin, S. G., and Benner, S. A. (2010) Expanded genetic alphabets in the polymerase chain reaction Angew. Chem., Int. Ed. 49, 177 DOI: 10.1002/anie.2009051739https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhs1Wlt7fP&md5=265d5ee46db9fd09fd0457ba6a471378Expanded genetic alphabets in the polymerase chain reactionYang, Zunyi; Chen, Fei; Chamberlin, Stephen G.; Benner, Steven A.Angewandte Chemie, International Edition (2010), 49 (1), 177-180, S177/1-S177/16CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)A flexible nested PCR using an artificially expanded genetic information system (AEGIS) was developed. A pyrimidine analog (Z) and its complementary purine analog (P) were used as extra genetic letters. Incorporated into DNA strands, they form Z:P pairs that contribute to duplex thermostability more than C:G pairs, discriminate against mismatches to the same extent as std. nucleobases, and are accepted by many polymerases, including Deep Vent and Taq.
- 10Yang, Z., Chen, F., Alvarado, J. B., and Benner, S. A. (2011) Amplification, mutation, and sequencing of a six-letter synthetic genetic system J. Am. Chem. Soc. 133, 15105 DOI: 10.1021/ja204910n10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtFaks7zL&md5=cc4561e9527ca359c82494deb9e0be2dAmplification, mutation, and sequencing of a six-letter synthetic genetic systemYang, Zun-Yi; Chen, Fei; Alvarado, J. Brian; Benner, Steven A.Journal of the American Chemical Society (2011), 133 (38), 15105-15112CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)The next goals in the development of a synthetic biol. that uses artificial genetic systems will require chem.-biol. combinations that allow the amplification of DNA contg. any no. of sequential and nonsequential nonstandard nucleotides. This amplification must ensure that the nonstandard nucleotides are not unidirectionally lost during PCR amplification (unidirectional loss would cause the artificial system to revert to an all-natural genetic system). Further, technol. is needed to sequence artificial genetic DNA mols. The work reported here meets all three of these goals for a six-letter artificially expanded genetic information system (AEGIS) that comprises four std. nucleotides (G, A, C, and T) and two addnl. nonstandard nucleotides (Z and P). We report polymerases and PCR conditions that amplify a wide range of GACTZP DNA sequences having multiple consecutive unnatural synthetic genetic components with low (0.2% per theor. cycle) levels of mutation. We demonstrate that residual mutation processes both introduce and remove unnatural nucleotides, allowing the artificial genetic system to evolve as such, rather than revert to a wholly natural system. We then show that mechanisms for these residual mutation processes can be exploited in a strategy to sequence "six-letter" GACTZP DNA. These are all not yet reported for any other synthetic genetic system.
- 11Georgiadis, M. M., Singh, I., Kellett, W. F., Hoshika, S., Benner, S. A., and Richards, N. G. (2015) Structural basis for a six nucleotide genetic alphabet J. Am. Chem. Soc. 137, 6947 DOI: 10.1021/jacs.5b0348211https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXot1SqtLc%253D&md5=243dac24bcf9c2a3b8e72c822eac7c74Structural Basis for a Six Nucleotide Genetic AlphabetGeorgiadis, Millie M.; Singh, Isha; Kellett, Whitney F.; Hoshika, Shuichi; Benner, Steven A.; Richards, Nigel G. J.Journal of the American Chemical Society (2015), 137 (21), 6947-6955CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Expanded genetic systems are most likely to work with natural enzymes if the added nucleotides pair with geometries that are similar to those displayed by std. duplex DNA. Here, we present crystal structures of 16-mer duplexes showing this to be the case with two nonstandard nucleobases (Z, 6-amino-5-nitro-2(1H)-pyridone and P, 2-amino-imidazo[1,2-a]-1,3,5-triazin-4(8H)one) that were designed to form a Z:P pair with a std. "edge on" Watson-Crick geometry, but joined by rearranged hydrogen bond donor and acceptor groups. One duplex, with four Z:P pairs, was crystd. with a reverse transcriptase host and adopts primarily a B-form. Another contained six consecutive Z:P pairs; it crystd. without a host in an A-form. In both structures, Z:P pairs fit canonical nucleobase hydrogen-bonding parameters and known DNA helical forms. Unique features include stacking of the nitro group on Z with the adjacent nucleobase ring in the A-form duplex. In both B- and A-duplexes, major groove widths for the Z:P pairs are approx. 1 Å wider than those of comparable G:C pairs, perhaps to accommodate the large nitro group on Z. Otherwise, ZP-rich DNA had many of the same properties as CG-rich DNA, a conclusion supported by CD studies in soln. The ability of std. duplexes to accommodate multiple and consecutive Z:P pairs is consistent with the ability of natural polymerases to biosynthesize those pairs. This, in turn, implies that the GACTZP synthetic genetic system can explore the entire expanded sequence space that addnl. nucleotides create, a major step forward in this area of synthetic biol.
- 12Sismour, A. M., Lutz, S., Park, J. H., Lutz, M. J., Boyer, P. L., Hughes, S. H., and Benner, S. A. (2004) PCR amplification of DNA containing non-standard base pairs by variants of reverse transcriptase from Human Immunodeficiency Virus-1 Nucleic Acids Res. 32, 728 DOI: 10.1093/nar/gkh241There is no corresponding record for this reference.
- 13Winiger, C. B., Kim, M. J., Hoshika, S., Shaw, R. W., Moses, J. D., Matsuura, M. F., Gerloff, D. L., and Benner, S. A. (2016) Polymerase Interactions with Wobble Mismatches in Synthetic Genetic Systems and Their Evolutionary Implications Biochemistry 55, 3847 DOI: 10.1021/acs.biochem.6b0053313https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtVGjtL%252FO&md5=80d5724b34f1228c5b0450f17f5f56c1Polymerase Interactions with Wobble Mismatches in Synthetic Genetic Systems and Their Evolutionary ImplicationsWiniger, Christian B.; Kim, Myong-Jung; Hoshika, Shuichi; Shaw, Ryan W.; Moses, Jennifer D.; Matsuura, Mariko F.; Gerloff, Dietlind L.; Benner, Steven A.Biochemistry (2016), 55 (28), 3847-3850CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)In addn. to completing the Watson- Crick nucleobase matching "concept" (big pairs with small, hydrogen bond donors pair with hydrogen bond acceptors), artificially expanded genetic information systems (AEGIS) also challenge DNA polymerases with a complete set of mismatches, including wobble mismatches. Here, we explore wobble mismatches with AEGIS with DNA polymerase 1 from Escherichia coli. Remarkably, we find that the polymerase tolerates an AEGIS:std. wobble that has the same geometry as the G:T wobble that polymerases have evolved to exclude but excludes a wobble geometry that polymerases have never encountered in natural history. These results suggest certain limits to "structural analogy" and "evolutionary guidance" as tools to help synthetic biologists expand DNA alphabets.
- 14Winiger, C. B., Shaw, R. W., Kim, M. J., Moses, J. D., Matsuura, M. F., and Benner, S. A. (2017) Expanded Genetic Alphabets: Managing Nucleotides That Lack Tautomeric, Protonated, or Deprotonated Versions Complementary to Natural Nucleotides ACS Synth. Biol. 6, 194 DOI: 10.1021/acssynbio.6b0019314https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhsFakurvO&md5=820723e07e1274af995b78de5bed483aExpanded Genetic Alphabets: Managing Nucleotides That Lack Tautomeric, Protonated, or Deprotonated Versions Complementary to Natural NucleotidesWiniger, Christian B.; Shaw, Ryan W.; Kim, Myong-Jung; Moses, Jennifer D.; Matsuura, Mariko F.; Benner, Steven A.ACS Synthetic Biology (2017), 6 (2), 194-200CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)2,4-Diaminopyrimidine (trivially K) and imidazo[1,2-a]-1,3,5-triazine-2(8H)-4(3H)-dione (trivially X) form a nucleobase pair with Watson-Crick geometry as part of an artificially expanded genetic information system (AEGIS). Unlike some other AEGIS components, neither K nor X have any minor tautomeric forms mispairing. In vivo and in vitro expts. show how DNA polymerase I from E. coli manages this geometric distortion in DNA with one K:X pair, and fails to manage it with two adjacent K:X pairs. In the analogous in vivo expt., E. coli lacking dKTP/dXTP cannot rescue chloramphenicol resistance from a plasmid contg. two adjacent K:X pairs. In addn. to illustrating how living bacteria manage unnatural nucleotides, these studies identify bacteria able to serve as selection environments for engineering cells that replicate AEGIS pairs.or any protonated-deprotonated states that are Watson-Crick complementary to any natural nucleobase. Thus, K:X pairs can be replaced by natural pairs during replication only by geometrically distorted base pairing that is not tolerated by either the polymerase or its repair mechanisms.
- 15Benner, S. A. and Shaw, R. W. In Vivo Conversion of Nucleosides in Plasmid DNA. US Patent Application 14218405, 2015.There is no corresponding record for this reference.
- 16Betz, K., Malyshev, D. A., Lavergne, T., Welte, W., Diederichs, K., Dwyer, T. J., Ordoukhanian, P., Romesberg, F. E., and Marx, A. (2012) KlenTaq polymerase replicates unnatural base pairs by inducing a Watson-Crick geometry Nat. Chem. Biol. 8, 612 DOI: 10.1038/nchembio.96616https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XnvVyrurw%253D&md5=3f53105607a419a1d1326125178d682dKlenTaq polymerase replicates unnatural base pairs by inducing a Watson-Crick geometryBetz, Karin; Malyshev, Denis A.; Lavergne, Thomas; Welte, Wolfram; Diederichs, Kay; Dwyer, Tammy J.; Ordoukhanian, Phillip; Romesberg, Floyd E.; Marx, AndreasNature Chemical Biology (2012), 8 (7), 612-614CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)Many candidate unnatural DNA base pairs have been developed, but some of the best-replicated pairs adopt intercalated structures in free DNA that are difficult to reconcile with known mechanisms of polymerase recognition. Here we present crystal structures of KlenTaq DNA polymerase at different stages of replication for one such pair, dNaM-d5SICS, and show that efficient replication results from the polymerase itself, inducing the required natural-like structure.
- 17Malyshev, D. A., Dhami, K., Lavergne, T., Chen, T., Dai, N., Foster, J. M., Correa, I. R., Jr., and Romesberg, F. E. (2014) A semi-synthetic organism with an expanded genetic alphabet Nature 509, 385 DOI: 10.1038/nature1331417https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXotVyqtb8%253D&md5=97b4b184cda52cc809b1705e5e88ad8eA semi-synthetic organism with an expanded genetic alphabetMalyshev, Denis A.; Dhami, Kirandeep; Lavergne, Thomas; Chen, Tingjian; Dai, Nan; Foster, Jeremy M.; Correa, Ivan R.; Romesberg, Floyd E.Nature (London, United Kingdom) (2014), 509 (7500), 385-388CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Organisms are defined by the information encoded in their genomes, and since the origin of life this information has been encoded using a two-base-pair genetic alphabet (A-T and G-C). In vitro, the alphabet has been expanded to include several unnatural base pairs (UBPs). We have developed a class of UBPs formed between nucleotides bearing hydrophobic nucleobases, exemplified by the pair formed between d5SICS and dNaM (d5SICS-dNaM), which is efficiently PCR-amplified and transcribed in vitro, and whose unique mechanism of replication has been characterized. However, expansion of an organism's genetic alphabet presents new and unprecedented challenges: the unnatural nucleoside triphosphates must be available inside the cell; endogenous polymerases must be able to use the unnatural triphosphates to faithfully replicate DNA contg. the UBP within the complex cellular milieu; and finally, the UBP must be stable in the presence of pathways that maintain the integrity of DNA. Here we show that an exogenously expressed algal nucleotide triphosphate transporter efficiently imports the triphosphates of both d5SICS and dNaM (d5SICSTP and dNaMTP) into Escherichia coli, and that the endogenous replication machinery uses them to accurately replicate a plasmid contg. d5SICS-dNaM. Neither the presence of the unnatural triphosphates nor the replication of the UBP introduces a notable growth burden. Lastly, we find that the UBP is not efficiently excised by DNA repair pathways. Thus, the resulting bacterium is the first organism to propagate stably an expanded genetic alphabet.
- 18Neidle, S. (1999) Nucleic Acid Structure, Oxford University Press, New York.There is no corresponding record for this reference.
- 19Molt, R. W., Jr., Georgiadis, M. M., and Richards, N. G. J. (2017) Consecutive non-natural PZ nucleobase pairs in DNA impact helical structure as seen in 50 mus molecular dynamics simulations Nucleic Acids Res. 45, 3643 DOI: 10.1093/nar/gkx144There is no corresponding record for this reference.
- 20Sun, D., Jessen, S., Liu, C., Liu, X., Najmudin, S., and Georgiadis, M. M. (1998) Cloning, expression, and purification of a catalytic fragment of Moloney murine leukemia virus reverse transcriptase: crystallization of nucleic acid complexes Protein Sci. 7, 1575 DOI: 10.1002/pro.556007071120https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXks1eqtLg%253D&md5=9e72f0850f5e4777c85e81a3a32f3074Cloning, expression, and purification of a catalytic fragment of Moloney murine leukemia virus reverse transcriptase: crystallization of nucleic acid complexesSun, Dunming; Jessen, Sven; Liu, Chunhui; Liu, Xiuping; Najmudin, Shabir; Georgiadis, Millie M.Protein Science (1998), 7 (7), 1575-1582CODEN: PRCIEI; ISSN:0961-8368. (Cambridge University Press)Reverse transcriptase is an essential retroviral enzyme that uses RNA- and DNA-directed DNA polymerase activities as well as an RNaseH activity to synthesize a double-stranded DNA copy of the single-stranded RNA genome. In an effort to obtain high-resoln. structural information regarding the polymerase active site of reverse transcriptase, we have pursued studies on a catalytic fragment from Moloney murine leukemia virus reverse transcriptase. DNA encoding the catalytic fragment, defined originally by limited proteolytic digestion, has been cloned, and the protein has been expressed and purified from Escherichia coli. The fragment obtained by limited proteolytic digestion and the bacterially expressed fragment retain polymerase activity. Crystn. studies involving nucleic acid complexes with a catalytic fragment from both sources are reported, including variables screened to improve crystals and cryocooling. Three crystal forms of catalytic fragment-nucleic acid complexes have been characterized, which all contain at least two protein mols. in the asym. unit. As isolated, the catalytic fragment is monomeric. This anal. indicates that the enzyme dimerizes in the presence of nucleic acid.
- 21Cote, M. L., Yohannan, S. J., and Georgiadis, M. M. (2000) Use of an N-terminal fragment from moloney murine leukemia virus reverse transcriptase to facilitate crystallization and analysis of a pseudo-16-mer DNA molecule containing G-A mispairs Acta Crystallogr., Sect. D: Biol. Crystallogr. 56, 1120 DOI: 10.1107/S0907444900008246There is no corresponding record for this reference.
- 22Najmudin, S., Cote, M. L., Sun, D., Yohannan, S., Montano, S. P., Gu, J., and Georgiadis, M. M. (2000) Crystal structures of an N-terminal fragment from Moloney murine leukemia virus reverse transcriptase complexed with nucleic acid: functional implications for template-primer binding to the fingers domain J. Mol. Biol. 296, 613 DOI: 10.1006/jmbi.1999.3477There is no corresponding record for this reference.
- 23Cote, M. L. and Georgiadis, M. M. (2001) Structure of a pseudo-16-mer DNA with stacked guanines and two G-A mispairs complexed with the N-terminal fragment of Moloney murine leukemia virus reverse transcriptase Acta Crystallogr., Sect. D: Biol. Crystallogr. 57, 1238 DOI: 10.1107/S090744490100943XThere is no corresponding record for this reference.
- 24Montano, S. P., Cote, M. L., Roth, M. J., and Georgiadis, M. M. (2006) Crystal structures of oligonucleotides including the integrase processing site of the Moloney murine leukemia virus Nucleic Acids Res. 34, 5353 DOI: 10.1093/nar/gkl693There is no corresponding record for this reference.
- 25Joyce, C. M. and Steitz, T. A. (1994) Function and structure relationships in DNA polymerases Annu. Rev. Biochem. 63, 777 DOI: 10.1146/annurev.bi.63.070194.00402125https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2MXitlCgtg%253D%253D&md5=5980e61ddb57aa9d5e0ad27a7c3aab61Function and structure relationships in DNA polymerasesJoyce, Catherine M.; Steitz, Thomas A.Annual Review of Biochemistry (1994), 63 (), 777-822CODEN: ARBOAW; ISSN:0066-4154.A review, with 184 refs., on the structural and mechanistic aspects of the reactions catalyzed by DNA polymerase.
- 26Hendrickson, C. L., Devine, K. G., and Benner, S. A. (2004) Probing minor groove recognition contacts by DNA polymerases and reverse transcriptases using 3-deaza-2′-deoxyadenosine Nucleic Acids Res. 32, 2241 DOI: 10.1093/nar/gkh542There is no corresponding record for this reference.
- 27El Hassan, M. a. and Calladine, C. R. (1998) Two distinct modes of protein-induced bending in DNA J. Mol. Biol. 282, 331 DOI: 10.1006/jmbi.1998.199427https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXmtlClsLc%253D&md5=f75d6fa540cc92d868bb129c3d110e22Two distinct modes of protein-induced bending in DNAEl Hassan, M. A.; Calladine, C. R.Journal of Molecular Biology (1998), 282 (2), 331-343CODEN: JMOBAK; ISSN:0022-2836. (Academic Press)Crystd. "naked" DNA oligomers in the B form show significant conformational mobility, particularly at CA/TG and TA/TA steps: there is a range in Roll angle of some 15° between consecutive base-pairs, and Slide and Twist are directly coupled to Roll. We call such motions "mode I". They are sufficient to enable DNA to curve gently around proteins such as histone octamers in the nucleosome particle. When DNA bends around other proteins, such as CAP and TBP, its distortion is much more severe. Although the DNA in close contact with these proteins includes the CA/TG and TA/TA steps, resp., the mode I flexibility is not deployed: instead, a more severe "mode II" manoeuvre is obsd. in DNA/protein co-crystals. Mode II has several distinctive phys. features. First, its range of Roll angle is much wider than for mode I. Second, the major-groove width remains more-or-less const. as Roll increases, whereas it decreases significantly as Roll increases in mode I; and this enables the major groove of the DNA to accommodate a protein moiety in its severely bent conformation. Third, the value of Slide remains more-or-less const. as Roll increases, whereas it decreases in mode I. In general, in both modes I and II, the major-groove width appears to be closely related to the Slide between base-pairs. In mode II there appears to be a definite "point pivot" on the major-groove side of the two base-pairs that constitute a dinucleotide step, formed either by the steric interlocking of propeller-twisted base-pairs or by a bifurcated hydrogen bond. Distortion of DNA in mode II seems to be an intrinsic property of the double-helical structure, since it occurs whether protein is bound on the major-groove side (e.g. CAP) or on the minor-groove side (e.g. TBP). Mode II distortion occurs in a wider range of steps than those that show the largest mode-I variation; nevertheless, "access" to mode II deformation appears to be gained via mode I distortion at particular steps CA/TG and TA/TA. (c) 1998 Academic Press.
- 28Lu, X. J. and Olson, W. K. (2003) 3DNA: a software package for the analysis, rebuilding and visualization of three-dimensional nucleic acid structures Nucleic Acids Res. 31, 5108 DOI: 10.1093/nar/gkg68028https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXmvVWrsLo%253D&md5=959e6f79a9a71e0ca7269c2736e6a4c33DNA: a software package for the analysis, rebuilding and visualization of three-dimensional nucleic acid structuresLu, Xiang-Jun; Olson, Wilma K.Nucleic Acids Research (2003), 31 (17), 5108-5121CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)We present a comprehensive software package, 3DNA, for the anal., reconstruction and visualization of three-dimensional nucleic acid structures. Starting from a coordinate file in Protein Data Bank (PDB) format, 3DNA can handle antiparallel and parallel double helixes, single-stranded structures, triplexes, quadruplexes and other complex tertiary folding motifs found in both DNA and RNA structures. The anal. routines identify and categorize all base interactions and classify the double helical character of appropriate base pair steps. The program makes use of a recently recommended ref. frame for the description of nucleic acid base pair geometry and a rigorous matrix-based scheme to calc. local conformational parameters and rebuild the structure from these parameters. The rebuilding routines produce rectangular block representations of nucleic acids as well as full at. models with the sugar-phosphate backbone and publication quality standardized' base stacking diagrams. Utilities are provided to locate the base pairs and helical regions in a structure and to reorient structures for effective visualization. Regular helical models based on x-ray diffraction measurements of various repeating sequences can also be generated within the program.
- 29Lu, X. J. and Olson, W. K. (2008) 3DNA: a versatile, integrated software system for the analysis, rebuilding and visualization of three-dimensional nucleic-acid structures Nat. Protoc. 3, 1213 DOI: 10.1038/nprot.2008.10429https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXotFaktbY%253D&md5=47da876b232ab75aaa6bda05fb6f10713DNA: a versatile, integrated software system for the analysis, rebuilding and visualization of three-dimensional nucleic-acid structuresLu, Xiang-Jun; Olson, Wilma K.Nature Protocols (2008), 3 (7), 1213-1227CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)The authors present a set of protocols showing how to use the 3DNA suite of programs to analyze, rebuild and visualize three-dimensional nucleic-acid structures. The software dets. a wide range of conformational parameters, including the identities and rigid-body parameters of interacting bases and base-pair steps, the nucleotides comprising helical fragments, the area of overlap of stacked bases and so on. The reconstruction of three-dimensional structure takes advantage of rigorously defined rigid-body parameters, producing rectangular block representations of the nucleic-acid bases and base pairs and all-atom models with approx. sugar-phosphate backbones. The visualization components create vector-based drawings and scenes that can be rendered as raster-graphics images, allowing for easy generation of publication-quality figures. The utility programs use geometric variables to control the view and scale of an object, for comparison of related structures. The commands run in seconds even for large structures. The software and related information are available at.
- 30Colasanti, A. V., Lu, X. J., and Olson, W. K. (2013) Analyzing and building nucleic acid structures with 3DNA J. Visualized Exp. e4401 DOI: 10.3791/4401There is no corresponding record for this reference.
- 31Prive, G. G., Heinemann, U., Chandrasegaran, S., Kan, L. S., Kopka, M. L., and Dickerson, R. E. (1987) Helix geometry, hydration, and G.A mismatch in a B-DNA decamer Science 238, 498 DOI: 10.1126/science.3310237There is no corresponding record for this reference.
- 32Grzeskowiak, K. (1996) Sequence-dependent structural variation in B-DNA Chem. Biol. 3, 785 DOI: 10.1016/S1074-5521(96)90062-932https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28Xmslyjtb8%253D&md5=cbaa4eb2c36acbed548746dac2b0d583Sequence-dependent structural variation in B-DNAGrzeskowiak, KazimierzChemistry & Biology (1996), 3 (10), 785-790CODEN: CBOLE2; ISSN:1074-5521. (Current Biology)A review and discussion with 38 refs. Although fiber diffraction originally led to the belief that the structure of DNA would be a simple regular helix, x-ray crystallog. of synthetic oligomers has shown that both deformability and structure depend on sequence. However, the rules that det. these factors remain mysterious.
- 33Berman, H. M. (1997) Crystal studies of B-DNA: the answers and the questions Biopolymers 44, 23 DOI: 10.1002/(SICI)1097-0282(1997)44:1<23::AID-BIP3>3.0.CO;2-1There is no corresponding record for this reference.
- 34Drew, H. R., Wing, R. M., Takano, T., Broka, C., Tanaka, S., Itakura, K., and Dickerson, R. E. (1981) Structure of a B-DNA dodecamer: conformation and dynamics Proc. Natl. Acad. Sci. U. S. A. 78, 2179 DOI: 10.1073/pnas.78.4.217934https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL3MXktVajt74%253D&md5=1278977a90de8b049e2577294d569c45Structure of a B-DNA dodecamer. I. Conformation and dynamicsDrew, Horace R.; Wing, Richard M.; Takano, Tsunehiro; Broka, Christopher; Tanaka, Shoji; Itakura, Keiichi; Dickerson, Richard E.Proceedings of the National Academy of Sciences of the United States of America (1981), 78 (4), 2179-83CODEN: PNASA6; ISSN:0027-8424.The crystal structure of the synthetic DNA dodecamer d(CpGpCpGpApApTpTpCpGpCpG) was refined to a residual error of R = 17.8% at 1.9-Å resoln. (2-σ data). The mol. forms slightly >1 complete turn of right-handed double-stranded B helix. The 2 ends of the helix overlap and interlock minor grooves with neighboring mols. up and down a 21 screw axis, producing a 19° bend in helix axis over the 11-base-pair steps of the dodecamer. In the center of the mol., where perturbation is least, the helix has a mean rotation of 36.9° per step, or 9.8 base pairs per turn. The mean propeller twist (total dihedral angle between base planes) between A·T base pairs in the center of the mol. is 17.3°, and that between C·G pairs on the 2 ends avs. 11.5°. Individual deoxyribose ring conformations, measured by the C5'-C4'-C3'-O3' torsion angle δ, exhibit an approx. Gaussian distribution centered around the C1'-exo position with δav. = 123° and a range of 79-157°. Purine sugars cluster at high δ values, and pyrimidine sugars cluster at lower δ. A tendency toward 2-fold symmetry in sugar conformation about the center of the mol. is detectable in spite of the destruction of ideal 2-fold symmetry by the mol. bending. More strikingly, sugar conformations of paired bases appear to follow a principle of anticorrelation, with δ values lying approx. the same distance to either side of the center value, δ = 123°. This same anticorrelation is also obsd. in other DNA and DNA·RNA structures.
- 35Sponer, J., Jurecka, P., and Hobza, P. (2004) Accurate interaction energies of hydrogen-bonded nucleic acid base pairs J. Am. Chem. Soc. 126, 10142 DOI: 10.1021/ja048436s35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXlvVOltr4%253D&md5=209861454eb2a02c4b70c08abb9abe25Accurate Interaction Energies of Hydrogen-Bonded Nucleic Acid Base PairsSponer, Jiri; Jurecka, Petr; Hobza, PavelJournal of the American Chemical Society (2004), 126 (32), 10142-10151CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Hydrogen-bonded nucleic acids base pairs substantially contribute to the structure and stability of nucleic acids. The study presents ref. ab initio structures and interaction energies of selected base pairs with binding energies ranging from -5 to -47 kcal/mol. The mol. structures are obtained using the RI-MP2 (resoln. of identity MP2) method with extended cc-pVTZ basis set of AOs. The RI-MP2 method provides results essentially identical with the std. MP2 method. The interaction energies are calcd. using the Complete Basis Set (CBS) extrapolation at the RI-MP2 level. For some base pairs, Coupled-Cluster corrections with inclusion of non-iterative triple contributions (CCSD(T)) are given. The calcns. are compared with selected medium quality methods. The PW91 DFT functional with the 6-31G** basis set matches well the RI-MP2/CBS abs. interaction energies and reproduces the relative values of base pairing energies with a max. relative error of 2.6 kcal/mol when applied with Becke3LYP-optimized geometries. The Becke3LYP DFT functional underestimates the interaction energies by few kcal/mol with relative error of 2.2 kcal/mol. Very good performance of nonpolarizable Cornell et al. force field is confirmed and this indirectly supports the view that H-bonded base pairs are primarily stabilized by electrostatic interactions.
- 36Bak, K. L., Jørgensen, P., Olsen, J., Helgaker, T., and Klopper, W. (2000) Accuracy of atomization energies and reaction enthalpies in standard and extrapolated electronic wave function/basis set calculations J. Chem. Phys. 112, 9229 DOI: 10.1063/1.48154436https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3cXjsVSnu7w%253D&md5=0f65812ac2c3cd8da5818cb1500f06f6Accuracy of atomization energies and reaction enthalpies in standard and extrapolated electronic wave function/basis set calculationsBak, Keld L.; Jorgensen, Poul; Olsen, Jeppe; Helgaker, Trygve; Klopper, WimJournal of Chemical Physics (2000), 112 (21), 9229-9242CODEN: JCPSA6; ISSN:0021-9606. (American Institute of Physics)The accuracy of std. ab initio wave function calcns. of atomization energies and reaction enthalpies has been assessed by comparing with exptl. data for 16 small closed-shell mols. and 13 isogyric reactions. The investigated wave function models are Hartree-Fock (HF), Moeller-Plesset second-order perturbation theory (MP2), coupled-cluster theory with singles and doubles excitations (CCSD) and CCSD with perturbative triple-excitation corrections [CCSD(T)]; the one-electron basis sets used are the correlation-consistent cc-pVxZ and cc-pCVxZ basis sets with cardinal nos. x = D, T, Q, 5, and 6. Results close to the basis-set limit have been obtained by using two-point extrapolations. In agreement with previous studies, it is found that the intrinsic error of the CCSD(T) method is less than chem. accuracy (≈4 kJ/mol) for both atomization energies and reaction enthalpies. The mean and max. abs. errors of the best CCSD(T) calcns. are 0.8 and 2.3 kJ/mol for the atomization energies and 1.0 and 2.3 kJ/mol for the reaction enthalpies. Chem. accuracy is obtained already from the extrapolations based on the cc-pCVTZ and cc-pCVQZ basis sets, with mean and max. abs. errors of 1.7 and 4.0 kJ/mol for atomization energies and 1.3 and 3.1 kJ/mol for reaction enthalpies. The intrinsic errors of the Hartree-Fock, MP2, and CCSD wave function models are significantly larger than for CCSD(T). For CCSD and MP2, the mean abs. errors in the basis set limit are about 32 kJ/mol for the atomization energies and about 10 and 15 kJ/mol, resp., for the reaction enthalpies. For the Hartree-Fock model, the mean abs. errors are 405 and 29 kJ/mol for atomization energies and reaction enthalpies, resp. Correlation of the core electrons is important in order to obtain accurate results with CCSD(T). Without compromising the accuracy, the core contribution may be calcd. with a basis set that has one cardinal no. lower than that used for the valence correlation contribution. Basis-set extrapolation should be used for both the core and the valence contributions.
- 37Watts, J. D., Gauss, J., and Bartlett, R. J. (1993) Coupled-cluster methods with noniterative triple excitations for restricted open-shell Hartree–Fock and other general single determinant reference functions. Energies and analytical gradients J. Chem. Phys. 98, 8718 DOI: 10.1063/1.46448037https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3sXksFKnsrY%253D&md5=af3b7d1ea8ac357af387d567a5f849c1Coupled-cluster methods with noniterative triple excitations for restricted-open-shell-Hartree-Fock and other general single-determinant reference functions. Energies and analytical gradientsWatts, John D.; Gauss, Juergen; Bartlett, Rodney J.Journal of Chemical Physics (1993), 98 (11), 8718-33CODEN: JCPSA6; ISSN:0021-9606.A new, noniterative triples correction to the CCSD method, for general single determinant ref. functions is proposed and investigated numerically for various cases, including non-Hartree-Fock (non-HF) ref. functions. It is correct through fourth-order of perturbation theory for non-HF refs., and unlike other such methods, retains the usual invariance properties common to CC methods, while requiring only a single N7 step. In the canonical Hartree-Fock case, the method is equiv. to the usual CCSD(T) method, but now permits the use of restricted open-shell Hartree-Fock (ROHF) and quasirestricted Hartree-Fock (QRHF) ref. determinants, along with many others. Comparisons with full CI (FCI) results are presented for CH2, CH2+, CH3, NH2, and SiH2. The derivation and initial computational implementation of anal. gradients for the ROHF-CCSD(T) method, which includes UHF (UHF) CCSD(T) and RHF-CCSD(T) as special cases, are also reported. Applications of anal. gradients are presented for HOO, the CN radical, which is highly spin contaminated at the UHF level, and HCO, the latter with several large basis sets. With these developments of anal. gradients, these highly accurate generalized CCSD(T) methods can be widely applied.
- 38Coester, F. and Kümmel, H. (1960) Short-Range Correlations in Nuclear Wave Functions Nucl. Phys. 17, 477 DOI: 10.1016/0029-5582(60)90140-138https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaF3MXmvFSlsw%253D%253D&md5=e99794469699b0b7595035ce600d6476Short-range correlations in nuclear wave functionsCoester, F.; Kummel, H.Nuclear Physics (1960), 17 (), 477-85CODEN: NUPHA7; ISSN:0029-5582.The 1- and 2-body d. matrixes are well approximated by expressions involving only the single-particle wave functions and the 2-body cluster functions. The Schr.ovrddot.odinger equation yielded a coupled set of equations which detd. the cluster functions as well as the single-particle wave functions.
- 39Purvis, G. D. and Bartlett, R. J. (1982) A full coupled-cluster singles and doubles model: The inclusion of disconnected triples J. Chem. Phys. 76, 1910 DOI: 10.1063/1.44316439https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL38XhtFSgtLY%253D&md5=0cf9405a65eed10485f6fc00646ef037A full coupled-cluster singles and doubles model: the inclusion of disconnected triplesPurvis, George D., III; Bartlett, Rodney J.Journal of Chemical Physics (1982), 76 (4), 1910-18CODEN: JCPSA6; ISSN:0021-9606.The coupled-cluster singles and doubles model (CCSD) is derived algebraically, presenting the full set of equations for a general ref. function explicitly in spin-orbital form. The computational implementation of the CCSD model, which involves cubic and quartic terms, is discussed and results are reported and compared with full CI calcns. for H2O and BeH2. The CCSD exponential ansatz sums higher-order correlation effects efficiently even for BeH2, near its transition state geometry where quasidegeneracy efforts are quite large, recovering 98% of the full CI correlation energy. For H2O, CCSD plus the 4th-order triple excitation correction agrees with the full CI energy to 0.5 kcal/mol. Comparisons with low-order models provide ests. of the effect of the higher-order terms on the correlation energy.
- 40Jorgensen, W. L. and Pranata, J. (1990) Importance of secondary interactions in triply hydrogen bonded complexes: guanine-cytosine vs uracil-2,6-diaminopyridine J. Am. Chem. Soc. 112, 2008 DOI: 10.1021/ja00161a06140https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK3cXhtFymsLc%253D&md5=2a10cf61aecf583270135a87f7c5adcbImportance of secondary interactions in triply hydrogen bonded complexes: guanine-cytosine vs uracil-2,6-diaminopyridineJorgensen, William L.; Pranata, JuliantoJournal of the American Chemical Society (1990), 112 (5), 2008-10CODEN: JACSAT; ISSN:0002-7863.Although complexes of guanine-cytosine and uracil-2,6-diaminopyridine are both triply H-bonded in CHCl3, measured assocn. consts. for such systems vary by 102-103. The origin of the discrepancy is analyzed here through computational studies. Monte Carlo statistical mechanics simulations for the complexes in CHCl3 also find the substantial binding preference for guanine-cytosine. The difference is then traced to the gas-phase interaction energies which favor guanine-cytosine complexation by ca. 10 kcal/mol. The three H bonds are of the same type in both complexes; however, the variation in their arrangement leads to secondary electrostatic effects that account for the destabilization of the uracil-2,6-diaminopyridine complex. Such secondary interactions are a significant element for consideration in mol. design.
- 41Wang, W., Sheng, X., Zhang, S., Huang, F., Sun, C., Liu, J., and Chen, D. (2016) Theoretical characterization of the conformational features of unnatural oligonucleotides containing a six nucleotide genetic alphabet Phys. Chem. Chem. Phys. 18, 28492 DOI: 10.1039/C6CP05594JThere is no corresponding record for this reference.
- 42Wang, X., Hoshika, S., Peterson, R. J., Kim, M. J., Benner, S. A., and Kahn, J. D. (2017) Biophysics of Artificially Expanded Genetic Information Systems. Thermodynamics of DNA Duplexes Containing Matches and Mismatches Involving 2-Amino-3-nitropyridin-6-one (Z) and Imidazo[1,2-a]-1,3,5-triazin-4(8H)one (P) ACS Synth. Biol. 6, 782 DOI: 10.1021/acssynbio.6b0022442https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtVKhtbg%253D&md5=8621b81e603d75d3079a67bf3768ae1bBiophysics of Artificially Expanded Genetic Information Systems. Thermodynamics of DNA Duplexes Containing Matches and Mismatches Involving 2-Amino-3-nitropyridin-6-one (Z) and Imidazo[1,2-a]-1,3,5-triazin-4(8H)one (P)Wang, Xiaoyu; Hoshika, Shuichi; Peterson, Raymond J.; Kim, Myong-Jung; Benner, Steven A.; Kahn, Jason D.ACS Synthetic Biology (2017), 6 (5), 782-792CODEN: ASBCD6; ISSN:2161-5063. (American Chemical Society)Synthetic nucleobases presenting non-Watson-Crick arrangements of hydrogen bond donor and acceptor groups can form addnl. nucleotide pairs that stabilize duplex DNA independent of the std. A:T and G:C pairs. The pair between 2-amino-3-nitropyridin-6-one 2'-deoxyriboside (presenting a {donor-donor-acceptor} hydrogen bonding pattern on the Watson-Crick face of the small component, trivially designated Z) and imidazo[1,2-a]-1,3,5-triazin-4(8H)one 2'-deoxyriboside (presenting an {acceptor-acceptor-donor} hydrogen bonding pattern on the large component, trivially designated P) is one of these extra pairs for which a substantial amt. of mol. biol. has been developed. Here, we report the results of UV absorbance melting measurements and det. the energetics of binding of DNA strands contg. Z and P to give short duplexes contg. Z:P pairs as well as various mismatches comprising Z and P. All measurements were done at 1 M NaCl in buffer (10 mM Na cacodylate, 0.5 mM EDTA, pH 7.0). Thermodn. parameters (ΔH°, ΔS°, and ΔG°37) for oligonucleotide hybridization were extd. Consistent with the Watson-Crick model that considers both geometric and hydrogen bonding complementarity, the Z:P pair was found to contribute more to duplex stability than any mismatches involving either nonstandard nucleotide. Further, the Z:P pair is more stable than a C:G pair. The Z:G pair was found to be the most stable mismatch, forming either a deprotonated mismatched pair or a wobble base pair analogous to the stable T:G mismatch. The C:P pair is less stable, perhaps analogous to the wobble pair obsd. for C:O6-methyl-G, in which the pyrimidine is displaced into the minor groove. The Z:A and T:P mismatches are much less stable. Parameters for predicting the thermodn. of oligonucleotides contg. Z and P bases are provided. This represents the first case where this has been done for a synthetic genetic system.
- 43Malyshev, D. A., Pfaff, D. A., Ippoliti, S. I., Hwang, G. T., Dwyer, T. J., and Romesberg, F. E. (2010) Solution structure, mechanism of replication, and optimization of an unnatural base pair Chem. - Eur. J. 16, 12650 DOI: 10.1002/chem.20100095943https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhtlCksrnI&md5=da5cf1e3c584834fe385ea153c05f7bcSolution Structure, Mechanism of Replication, and Optimization of an Unnatural Base PairMalyshev, Denis A.; Pfaff, Danielle A.; Ippoliti, Shannon I.; Hwang, Gil Tae; Dwyer, Tammy J.; Romesberg, Floyd E.Chemistry - A European Journal (2010), 16 (42), 12650-12659, S12650/1-S12650/28CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)As part of an ongoing effort to expand the genetic alphabet for in vitro and eventual in vivo applications, we have synthesized a wide variety of predominantly hydrophobic unnatural base pairs and evaluated their replication in DNA. Collectively, the results have led us to propose that these base pairs, which lack stabilizing edge-on interactions, are replicated by means of a unique intercalative mechanism. Here, we report the synthesis and characterization of three novel derivs. of the nucleotide analog dMMO2, which forms an unnatural base pair with the nucleotide analog d5SICS. Replacing the para-Me substituent of dMMO2 with an annulated furan ring (yielding dFMO) has a dramatically neg. effect on replication, while replacing it with a methoxy (dDMO) or with a thiomethyl group (dTMO) improves replication in both steady-state assays and during PCR amplification. Thus, dTMO-d5SICS, and esp. dDMO-d5SICS, represent significant progress toward the expansion of the genetic alphabet. To elucidate the structure-activity relationships governing unnatural base pair replication, we detd. the soln. structure of duplex DNA contg. the parental dMMO2-d5SICS pair, and also used this structure to generate models of the deriv. base pairs. The results strongly support the intercalative mechanism of replication, reveal a surprisingly high level of specificity that may be achieved by optimizing packing interactions, and should prove invaluable for the further optimization of the unnatural base pair.
- 44Kimoto, M., Kawai, R., Mitsui, T., Yokoyama, S., and Hirao, I. (2009) An unnatural base pair system for efficient PCR amplification and functionalization of DNA molecules Nucleic Acids Res. 37, e14 DOI: 10.1093/nar/gkn95644https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtlGksL8%253D&md5=8812c6b48c1a2c611efdd4f77afda742An unnatural base pair system for efficient PCR amplification and functionalization of DNA moleculesKimoto, Michiko; Kawai, Rie; Mitsui, Tsuneo; Yokoyama, Shigeyuki; Hirao, IchiroNucleic Acids Research (2009), 37 (2), e14/1-e14/9CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)Toward the expansion of the genetic alphabet, we present an unnatural base pair system for efficient PCR amplification, enabling the site-specific incorporation of extra functional components into DNA. This system can be applied to conventional PCR protocols employing DNA templates contg. unnatural bases, natural and unnatural base triphosphates, and a 3'→5' exonuclease-proficient DNA polymerase. For highly faithful and efficient PCR amplification involving the unnatural base pairing, we identified the natural-base sequences surrounding the unnatural bases in DNA templates by an in vitro selection technique, using a DNA library contg. the unnatural base. The system facilitates the site-specific incorporation of a variety of modified unnatural bases, linked with functional groups of interest, into amplified DNA. DNA fragments (0.15 amol) contg. the unnatural base pair can be amplified 107-fold by 30 cycles of PCR, with <1% total mutation rate of the unnatural base pair site. Using the system, we demonstrated efficient PCR amplification and functionalization of DNA fragments for the extremely sensitive detection of zeptomol-scale target DNA mols. from mixts. with excess amts. (pmol scale) of foreign DNA species. This unnatural base pair system will be applicable to a wide range of DNA/RNA-based technologies.
- 45Yamashige, R., Kimoto, M., Takezawa, Y., Sato, A., Mitsui, T., Yokoyama, S., and Hirao, I. (2012) Highly specific unnatural base pair systems as a third base pair for PCR amplification Nucleic Acids Res. 40, 2793 DOI: 10.1093/nar/gkr106845https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XkvFOku70%253D&md5=cb29171678a521654ea4966f14365018Highly specific unnatural base pair systems as a third base pair for PCR amplificationYamashige, Rie; Kimoto, Michiko; Takezawa, Yusuke; Sato, Akira; Mitsui, Tsuneo; Yokoyama, Shigeyuki; Hirao, IchiroNucleic Acids Research (2012), 40 (6), 2793-2806CODEN: NARHAD; ISSN:0305-1048. (Oxford University Press)Toward the expansion of the genetic alphabet of DNA, we present highly efficient unnatural base pair systems as an artificial third base pair for PCR. Hydrophobic unnatural base pair systems between 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) and 2-nitro-4-propynylpyrrole (Px) were fine-tuned for efficient PCR, by assessing the amplification efficiency and fidelity using different polymerases and template sequence contexts and modified Px bases. Then, we found that some modifications of the Px base reduced the misincorporation rate of the unnatural base substrates opposite the natural bases in templates without reducing the Ds-Px pairing selectivity. Under optimized conditions using Deep Vent DNA polymerase, the misincorporation rate was extremely low (0.005%/bp/replication), which is close to that of the natural base mispairings by the polymerase. DNA fragments with different sequence contexts were amplified ∼1010-fold by 40 cycles of PCR, and the selectivity of the Ds-Px pairing was >99.9%/replication, except for 99.77%/replication for unfavorable purine-Ds-purine motifs. Furthermore, >97% of the Ds-Px pair in DNA survived in the 1028-fold amplified products after 100-cycle PCR (10 cycles repeated 10 times). This highly specific Ds-Px pair system provides a framework for new biotechnol.
- 46Betz, K., Kimoto, M., Diederichs, K., Hirao, I., and Marx, A. (2017) Structural Basis for Expansion of the Genetic Alphabet with an Artificial Nucleobase Pair Angew. Chem., Int. Ed. DOI: 10.1002/anie.201706478There is no corresponding record for this reference.
- 47Kirnos, M. D., Khudyakov, I. Y., Alexandrushkina, N. I., and Vanyushin, B. F. (1977) 2-aminoadenine is an adenine substituting for a base in S-2L cyanophage DNA Nature 270, 369 DOI: 10.1038/270369a047https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaE1cXhtVWhtbk%253D&md5=cda382dcaf4600a055736d1c2daf6d252-Aminoadenine is an adenine substituting for a base in S-2L cyanophage DNAKirnos, M. D.; Khudyakov, I. Y.; Alexandrushkina, N. I.; Vanyushin, B. F.Nature (London, United Kingdom) (1977), 270 (5635), 369-70CODEN: NATUAS; ISSN:0028-0836.Full enzymic hydrolysis of DNA from cyanophage S-2L revealed that deoxyadenine was completely substituted for by 2-aminoadenine deoxyribonucleotide (I). The DNA contained I, deoxythymidine, deoxycytidine, and deoxyguanidine in molar percentages 15.9, 15.4, 34.4, and 3.43, resp. The addnl. amino group offered by I permits the formation of 3 H-bonds in the I-thymidine base pair; this accounts for the higher Tm of 85.6° for the I-contg. S-2L DNA compared with 82.0° for adenine-contg. DNA.
- 48Howard, F. B. and Miles, H. T. (1984) 2NH2A X T helices in the ribo- and deoxypolynucleotide series. Structural and energetic consequences of 2NH2A substitution Biochemistry 23, 6723 DOI: 10.1021/bi00321a068There is no corresponding record for this reference.
- 49Minor, W., Cymborowski, M., Otwinowski, Z., and Chruszcz, M. (2006) HKL-3000: the integration of data reduction and structure solution--from diffraction images to an initial model in minutes Acta Crystallogr., Sect. D: Biol. Crystallogr. 62, 859 DOI: 10.1107/S090744490601994949https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XntVWksL8%253D&md5=2cd2fe288f954837abb4acae4c008a2fHKL-3000: the integration of data reduction and structure solution - from diffraction images to an initial model in minutesMinor, Wladek; Cymborowski, Marcin; Otwinowski, Zbyszek; Chruszcz, MaksymilianActa Crystallographica, Section D: Biological Crystallography (2006), D62 (8), 859-866CODEN: ABCRE6; ISSN:0907-4449. (Blackwell Publishing Ltd.)A new approach that integrates data collection, data redn., phasing and model building significantly accelerates the process of structure detn. and on av. minimizes the no. of data sets and synchrotron time required for structure soln. Initial testing of the HKL-3000 system (the beta version was named HKL-2000_ph) with more than 140 novel structure detns. has proven its high value for MAD/SAD expts. The heuristics for choosing the best computational strategy at different data resoln. limits of phasing signal and crystal diffraction are being optimized. The typical end result is an interpretable electron-d. map with a partially built structure and, in some cases, an almost complete refined model. The current development is oriented towards very fast structure soln. in order to provide feedback during the diffraction expt. Work is also proceeding towards improving the quality of phasing calcn. and model building.
- 50Vagin, A. and Teplyakov, A. (2010) Molecular replacement with MOLREP Acta Crystallogr., Sect. D: Biol. Crystallogr. 66, 22 DOI: 10.1107/S090744490904258950https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXit1Kktw%253D%253D&md5=820d114719aca209994ffb0403e3b20dMolecular replacement with MOLREPVagin, Alexei; Teplyakov, AlexeiActa Crystallographica, Section D: Biological Crystallography (2010), 66 (1), 22-25CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)MOLREP is an automated program for mol. replacement that utilizes a no. of original approaches to rotational and translational search and data prepn. Since the first publication describing the program, MOLREP has acquired a variety of features that include weighting of the X-ray data and search models, multi-copy search, fitting the model into electron d., structural superposition of two models and rigid-body refinement. The program can run in a fully automatic mode using optimized parameters calcd. from the input data.
- 51Emsley, P., Lohkamp, B., Scott, W. G., and Cowtan, K. (2010) Features and development of Coot Acta Crystallogr., Sect. D: Biol. Crystallogr. 66, 486 DOI: 10.1107/S090744491000749351https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXksFKisb8%253D&md5=67262cbfc60004de5ef962d5c043c910Features and development of CootEmsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K.Acta Crystallographica, Section D: Biological Crystallography (2010), 66 (4), 486-501CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)Coot is a mol.-graphics application for model building and validation of biol. macromols. The program displays electron-d. maps and at. models and allows model manipulations such as idealization, real-space refinement, manual rotation/translation, rigid-body fitting, ligand search, solvation, mutations, rotamers and Ramachandran idealization. Furthermore, tools are provided for model validation as well as interfaces to external programs for refinement, validation and graphics. The software is designed to be easy to learn for novice users, which is achieved by ensuring that tools for common tasks are 'discoverable' through familiar user-interface elements (menus and toolbars) or by intuitive behavior (mouse controls). Recent developments have focused on providing tools for expert users, with customisable key bindings, extensions and an extensive scripting interface. The software is under rapid development, but has already achieved very widespread use within the crystallog. community. The current state of the software is presented, with a description of the facilities available and of some of the underlying methods employed.
- 52Murshudov, G. N., Vagin, A. A., and Dodson, E. J. (1997) Refinement of macromolecular structures by the maximum-likelihood method Acta Crystallogr., Sect. D: Biol. Crystallogr. 53, 240 DOI: 10.1107/S090744499601225552https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXjs1Gnsb4%253D&md5=ec7f141ce1542f7ff458b98ecfe3f8afRefinement of macromolecular structures by the maximum-likelihood methodMurshudov, Garib N.; Vagin, Alexei A.; Dodson, Eleanor J.Acta Crystallographica, Section D: Biological Crystallography (1997), D53 (3), 240-255CODEN: ABCRE6; ISSN:0907-4449. (Munksgaard)A review with many refs. on the math. basis of max. likelihood. The likelihood function for macromol. structures is extended to include prior phase information and exptl. std. uncertainties. The assumption that different parts of a structure might have different errors is considered. A method for estg. σA using "free" reflections is described and its effects analyzed. The derived equations have been implemented in the program REFMAC. This has been tested on several proteins at different stages of refinement (bacterial α-amylase, cytochrome c', cross-linked insulin and oligopeptide binding protein). The results derived using the max.-likelihood residual are consistently better than those obtained from least-squares refinement.
- 53Adams, P. D., Afonine, P. V., Bunkoczi, G., Chen, V. B., Davis, I. W., Echols, N., Headd, J. J., Hung, L. W., Kapral, G. J., Grosse-Kunstleve, R. W., McCoy, A. J., Moriarty, N. W., Oeffner, R., Read, R. J., Richardson, D. C., Richardson, J. S., Terwilliger, T. C., and Zwart, P. H. (2010) PHENIX: a comprehensive Python-based system for macromolecular structure solution Acta Crystallogr., Sect. D: Biol. Crystallogr. 66, 213 DOI: 10.1107/S090744490905292553https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhs1Sisbc%253D&md5=67b439ff4bd61c659cae37ca4209b7bcPHENIX: a comprehensive Python-based system for macromolecular structure solutionAdams, Paul D.; Afonine, Pavel V.; Bunkoczi, Gabor; Chen, Vincent B.; Davis, Ian W.; Echols, Nathaniel; Headd, Jeffrey J.; Hung, Li Wei; Kapral, Gary J.; Grosse-Kunstleve, Ralf W.; McCoy, Airlie J.; Moriarty, Nigel W.; Oeffner, Robert; Read, Randy J.; Richardson, David C.; Richardson, Jane S.; Terwilliger, Thomas C.; Zwart, Peter H.Acta Crystallographica, Section D: Biological Crystallography (2010), 66 (2), 213-221CODEN: ABCRE6; ISSN:0907-4449. (International Union of Crystallography)A review. Macromol. X-ray crystallog. is routinely applied to understand biol. processes at a mol. level. However, significant time and effort are still required to solve and complete many of these structures because of the need for manual interpretation of complex numerical data using many software packages and the repeated use of interactive three-dimensional graphics. PHENIX has been developed to provide a comprehensive system for macromol. crystallog. structure soln. with an emphasis on the automation of all procedures. This has relied on the development of algorithms that minimize or eliminate subjective input, the development of algorithms that automate procedures that are traditionally performed by hand and, finally, the development of a framework that allows a tight integration between the algorithms.
- 54Zhao, Y. and Truhlar, D. G. (2008) The M06 suite of density functionals for main group thermochemistry, thermochemical kinetics, noncovalent interactions, excited states, and transition elements: two new functionals and systematic testing of four M06-class functionals and 12 other functionals Theor. Chem. Acc. 120, 215 DOI: 10.1007/s00214-007-0310-x54https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXltFyltbY%253D&md5=c31d6f319d7c7a45aa9b716220e4a422The M06 suite of density functionals for main group thermochemistry, thermochemical kinetics, noncovalent interactions, excited states, and transition elements: two new functionals and systematic testing of four M06-class functionals and 12 other functionalsZhao, Yan; Truhlar, Donald G.Theoretical Chemistry Accounts (2008), 120 (1-3), 215-241CODEN: TCACFW; ISSN:1432-881X. (Springer GmbH)We present two new hybrid meta exchange-correlation functionals, called M06 and M06-2X. The M06 functional is parametrized including both transition metals and nonmetals, whereas the M06-2X functional is a high-nonlocality functional with double the amt. of nonlocal exchange (2X), and it is parametrized only for nonmetals. The functionals, along with the previously published M06-L local functional and the M06-HF full-Hartree-Fock functionals, constitute the M06 suite of complementary functionals. We assess these four functionals by comparing their performance to that of 12 other functionals and Hartree-Fock theory for 403 energetic data in 29 diverse databases, including ten databases for thermochem., four databases for kinetics, eight databases for noncovalent interactions, three databases for transition metal bonding, one database for metal atom excitation energies, and three databases for mol. excitation energies. We also illustrate the performance of these 17 methods for three databases contg. 40 bond lengths and for databases contg. 38 vibrational frequencies and 15 vibrational zero point energies. We recommend the M06-2X functional for applications involving main-group thermochem., kinetics, noncovalent interactions, and electronic excitation energies to valence and Rydberg states. We recommend the M06 functional for application in organometallic and inorganometallic chem. and for noncovalent interactions.
- 55Kendall, R. A., Dunning, T. H., Jr, and Harrison, R. J. (1992) Electron affinities of the first-row atoms revisited. Systematic basis sets and wave functions J. Chem. Phys. 96, 6796 DOI: 10.1063/1.46256955https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK38XktFClurw%253D&md5=948a06eee10604a8fa37eae2b2ada4beElectron affinities of the first-row atoms revisited. Systematic basis sets and wave functionsKendall, Rick A.; Dunning, Thom H., Jr.; Harrison, Robert J.Journal of Chemical Physics (1992), 96 (9), 6796-806CODEN: JCPSA6; ISSN:0021-9606.The authors describe a reliable procedure for calcg. the electron affinity of an atom and present results for H, B, C, O, and F (H is included for completeness). This procedure involves the use of the recently proposed correlation-consistent basis sets augmented with functions to describe the more diffuse character of the at. anion coupled with a straightforward, uniform expansion of the ref. space for multireference singles and doubles configuration-interaction (MRSD-CI) calcns. A comparison is given with previous results and with corresponding full CI calcns. The most accurate EAs obtained from the MRSD-CI calcns. are (with exptl. values in parentheses): H 0.740 eV (0.754), B 0.258 (0.277), C 1.245 (1.263), O 1.384 (1.461), and F 3.337 (3.401). The EAs obtained from the MR-SDCI calcns. differ by less than 0.03 eV from those predicted by the full CI calcns.
- 56Frisch, M. J., Trucks, G. W., Schlegel, H. B., Scuseria, G. E., Robb, M. A., Cheeseman, J. R., Scalmani, G., Barone, V., Mennucci, B., Petersson, G. A., Nakatsuji, H., Caricato, M., Li, X., Hratchian, H. P., Izmaylov, A. F., Bloino, J., Zheng, G., Sonnenberg, J. L., Hada, M., Ehara, M., Toyota, K., Fukuda, R., Hasegawa, J., Ishida, M., Nakajima, T., Honda, Y., Kitao, O., Nakai, H., Vreven, T., Montgomery, J. A., Jr., Peralta, J. E., Ogliaro, F., Bearpark, M., Heyd, J. J., Brothers, E., Kudin, K. N., Staroverov, V. N., Kobayashi, R., Normand, J., Raghavachari, K., Rendell, A., Burant, J. C., Iyengar, S. S., Tomasi, J., Cossi, M., Rega, N., Millam, J. M., Klene, M., Knox, J. E., Cross, J. B., Bakken, V., Adamo, C., Jaramillo, J., Gomperts, R., Stratmann, R. E., Yazyev, O., Austin, A. J., Cammi, R., Pomelli, C., Ochterski, J. W., Martin, R. L., Morokuma, K., Zakrzewski, V. G., Voth, G. A., Salvador, P., Dannenberg, J. J., Dapprich, S., Daniels, A. D., Farkas, O., Foresman, J. B., Ortiz, J. V., Cioslowski, J., and Fox, D. J. (2009) Gaussian 09, revision E.01, Gaussian, Inc., Wallingford, CT.There is no corresponding record for this reference.
- 57Hohenberg, P. and Kohn, W. (1964) Inhomogeneous Electron Gas Phys. Rev. 136, B864 DOI: 10.1103/PhysRev.136.B864There is no corresponding record for this reference.
- 58Kohn, W. and Sham, L. J. (1965) Self-Consistent Equations Including Exchange and Correlation Effects Phys. Rev. 140, A1133 DOI: 10.1103/PhysRev.140.A1133There is no corresponding record for this reference.
- 59Řezáč, J. and Hobza, P. (2016) Benchmark Calculations of Interaction Energies in Noncovalent Complexes and Their Applications Chem. Rev. 116, 5038 DOI: 10.1021/acs.chemrev.5b0052659https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xjs1yqsb4%253D&md5=09e9bdc03605dac8bc2054321c71480dBenchmark Calculations of Interaction Energies in Noncovalent Complexes and Their ApplicationsRezac, Jan; Hobza, PavelChemical Reviews (Washington, DC, United States) (2016), 116 (9), 5038-5071CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. Data sets of benchmark interaction energies in noncovalent complexes are an important tool for quantifying the accuracy of computational methods used in this field, as well as for the development of new computational approaches. This review is intended as a guide to conscious use of these data sets. We discuss their construction and accuracy, list the data sets available in the literature, and demonstrate their application to validation and parametrization of quantum-mech. computational methods. In practical model systems, the benchmark interaction energies are usually obtained using composite CCSD(T)/CBS schemes. To use these results as a benchmark, their accuracy should be estd. first. We analyze the errors of this methodol. with respect to both the approxns. involved and the basis set size. We list the most prominent data sets covering various aspects of the field, from general ones to sets focusing on specific types of interactions or systems. The benchmark data are then used to validate more efficient computational approaches, including those based on explicitly correlated methods. Special attention is paid to the transition to large systems, where accurate benchmarking is difficult or impossible, and to the importance of nonequil. geometries in parametrization of more approx. methods.
- 60Lotrich, V. F., Flocke, N., Ponton, M., Yau, A. D., Perera, A. S., Deumens, E., and Bartlett, R. J. (2008) Parallel implementation of electronic structure energy, gradient, and Hessian calculations J. Chem. Phys. 128, 194104 DOI: 10.1063/1.292048260https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXmt12qtL0%253D&md5=007f341da25be49d1aed06ecf0db15e4Parallel implementation of electronic structure energy, gradient, and Hessian calculationsLotrich, V.; Flocke, N.; Ponton, M.; Yau, A. D.; Perera, A.; Deumens, E.; Bartlett, R. J.Journal of Chemical Physics (2008), 128 (19), 194104/1-194104/15CODEN: JCPSA6; ISSN:0021-9606. (American Institute of Physics)ACES III is a newly written program in which the computationally demanding components of the computational chem. code ACES II; [ACES II program system, University of Florida, 1994] have been redesigned and implemented in parallel. The high-level algorithms include Hartree-Fock (HF) SCF, second-order many-body perturbation theory [MBPT(2)] energy, gradient, and Hessian, and coupled cluster singles, doubles, and perturbative triples [CCSD(T)] energy and gradient. For SCF, MBPT(2), and CCSD(T), both restricted HF and unrestricted HF ref. wave functions are available. For MBPT(2) gradients and Hessians, a restricted open-shell HF ref. is also supported. The methods are programmed in a special language designed for the parallelization project. The language is called super instruction assembly language (SIAL). The design uses an extreme form of object-oriented programing. All compute intensive operations, such as tensor contractions and diagonalizations, all communication operations, and all input-output operations are handled by a parallel program written in C and FORTRAN 77. This parallel program, called the super instruction processor (SIP), interprets and executes the SIAL program. By sepg. the algorithmic complexity (in SIAL) from the complexities of execution on computer hardware (in SIP), a software system is created that allows for very effective optimization and tuning on different hardware architectures with quite manageable effort. (c) 2008 American Institute of Physics.
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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssynbio.7b00150.
Figure S1. UV absorbance as a function of pH for the dX nucleoside gives pKa of 8.5 ± 0.1. Table S1. Melting point temperatures for oligonucleotides containing X:K pairs Coordinates of M06-2X/aug″-cc-pVDZ optimized structures, Å (PDF)
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