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In Vivo Structure–Activity Relationships and Optimization of an Unnatural Base Pair for Replication in a Semi-Synthetic Organism
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In Vivo Structure–Activity Relationships and Optimization of an Unnatural Base Pair for Replication in a Semi-Synthetic Organism
体内研究结构-活性关系和非天然碱基对的优化，以便在半合成生物体中复制
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Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, United States
*floyd@scripps.edu
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Journal of the American Chemical Society

Cite this: J. Am. Chem. Soc. 2017, 139, 33, 11427–11433
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https://doi.org/10.1021/jacs.7b03540
Published August 10, 2017
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Abstract 抽象

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In an effort to expand the genetic alphabet and create semi-synthetic organisms (SSOs) that store and retrieve increased information, we have developed the unnatural base pairs (UBPs) dNaM and d5SICS or dTPT3 (dNaM-d5SICS and dNaM-dTPT3). The UBPs form based on hydrophobic and packing forces, as opposed to complementary hydrogen bonding, and while they are both retained within the in vivo environment of an Escherichia coli SSO, their development was based on structure–activity relationship (SAR) data generated in vitro. To address the likely possibility of different requirements of the in vivo environment, we screened 135 candidate UBPs for optimal performance in the SSO. Interestingly, we find that in vivo SARs differ from those collected in vitro, and most importantly, we identify four UBPs whose retention in the DNA of the SSO is higher than that of dNaM-dTPT3, which was previously the most promising UBP identified. The identification of these four UBPs further demonstrates that when optimized, hydrophobic and packing forces may be used to replace the complementary hydrogen bonding used by natural pairs and represents a significant advance in our continuing efforts to develop SSOs that store and retrieve more information than natural organisms.
为了扩展遗传字母表并创造存储和检索更多信息的半合成生物体 （SSO），我们开发了非天然碱基对 （UBP） dNaM 和 d5SICS 或 dTPT3（dNaM-d 5SICS 和 dNaM-d TPT3）。UBP 基于疏水力和堆积力形成，而不是互补氢键，虽然它们都保留在大肠杆菌 SSO 的体内环境中，但它们的开发是基于体外生成的构效关系 （SAR） 数据。为了满足体内环境可能出现的不同要求，我们筛选了 135 个候选 UBP，以便在 SSO 中实现最佳性能。有趣的是，我们发现体内 SARs 与体外收集的 SAR 不同，最重要的是，我们确定了四个 UBP，它们在 SSO DNA 中的保留率高于 dNaM-d TPT3，后者是以前发现的最有希望的 UBP。这四种 UBP 的鉴定进一步表明，优化后，疏水力和堆积力可用于替代自然对使用的互补氢键，这代表了我们不断努力开发比自然生物体存储和检索更多信息的 SSO 的重大进步。

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Introduction 介绍

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Natural organisms store genetic information in an alphabet of four nucleotide “letters,” and the replication and retrieval of this information is mediated by their selective pairing to form two base pairs. The addition of two new letters that form an unnatural base pair (UBP) would increase the potential information content and lay the foundation for semi-synthetic organisms (SSOs) capable of producing proteins with unnatural amino acids or even possessing novel forms and functions, which is the central goal of synthetic biology. (1) We, (2) as well as the Benner (3) and Hirao (4) groups, have worked toward this goal and have each identified UBPs that are well replicated in vitro. Our efforts have focused upon a family of UBPs represented by dNaM-d5SICS and dNaM-dTPT3 (Figure 1), which utilize hydrophobic and packing forces, as opposed to complementary hydrogen bonding, to stably pair within duplex DNA and during replication. (5-10) This mode of pairing results in an edge-to-edge, Watson–Crick-like geometry during unnatural triphosphate insertion within the polymerase active site, but cross-strand intercalation once the UBP is synthesized, (11-13) which likely mandates deintercalation for continued synthesis. Thus, the extended aromatic surface area of the unnatural nucleobases may be a liability. Nonetheless, with the unnatural triphosphates imported via transgenic expression of the nucleoside triphosphate transporter PtNTT2, we developed an Escherichia coli SSO that stably retains these UBPs in its DNA. (10, 14) While the retention of the UBPs in the DNA of the SSO is not equivalent to that of a natural base pair, retention may be raised to natural-like levels through the action of Cas9 targeted to degrade the DNA that has lost the UBP. (14)
自然生物体将遗传信息存储在由四个核苷酸“字母”组成的字母表中，这些信息的复制和检索是通过它们的选择性配对形成两个碱基对来介导的。添加两个形成非天然碱基对 （UBP） 的新字母将增加潜在的信息内容，并为半合成生物 （SSO） 奠定基础，这些生物体能够产生具有非天然氨基酸的蛋白质，甚至具有新的形式和功能，这是合成生物学的中心目标。（1） 我们 （2） 以及 Benner （3） 和 Hirao （4） 小组都朝着这个目标努力，并各自确定了在体外良好复制的 UBP。我们的工作集中在以 dNaM-d 5SICS 和 dNaM-d TPT3 为代表的 UBP 家族（图 1），它们利用疏水和堆积力，而不是互补氢键，在双链 DNA 内和复制过程中稳定配对。（5-10） 这种配对模式在聚合酶活性位点内不自然地插入三磷酸盐时产生无边的 Watson-Crick 样几何形状，但一旦合成 UBP，就会出现交叉链插层，（11-13） 这可能要求脱嵌以继续合成。因此，非天然核碱基的扩展芳香族表面积可能是一种负担。尽管如此，随着通过核苷三磷酸转运蛋白 PtNTT2 的转基因表达输入的非天然三磷酸盐，我们开发了一种大肠杆菌 SSO，它可以将这些 UBP 稳定地保留在其 DNA 中。 （10， 14）虽然 UBP 在 SSO DNA 中的保留率不等同于天然碱基对的保留率，但可以通过 Cas9 的作用将保留率提高到天然水平，从而降解丢失 UBP 的 DNA。（14）

Figure 1 图 1

Figure 1. The dNaM-d5SICS and dNaM-dTPT3 UBPs.
图 1.dNaM-d 5SICS 和 dNaM-d TPT3 UBP。

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Both the dNaM-d5SICS and dNaM-dTPT3 UBPs were identified from an intensive investigation of over 150 analogs that were evaluated in vitro, initially via steady-state kinetics and later by retention in PCR-amplified DNA. (2) These studies provided the key structure–activity relationship (SAR) data used to optimize the unnatural nucleotides, but the in vivo environment of the SSO introduces additional constraints, such as toxicity, import, and polymerase availability. Thus, it is unclear whether UBPs optimized based on in vitro SAR are optimal for performance in vivo. Indeed, during in vitro optimization, we emphasized the importance of identifying multiple different UBPs whose constituent nucleotides possess varying physicochemical properties to provide flexibility during the effort to deploy them in an SSO. (8, 15)
dNaM-d 5SICS 和 dNaM-d TPT3 UBP 都是通过对 150 多种类似物的深入调查鉴定的，这些类似物在体外进行了评估，最初是通过稳态动力学，后来是通过保留在 PCR 扩增的 DNA 中。（2） 这些研究提供了用于优化非天然核苷酸的关键构效关系 （SAR） 数据，但 SSO 的体内环境引入了额外的限制，例如毒性、输入和聚合酶可用性。因此，目前尚不清楚基于体外 SAR 优化的 UBP 是否最适合体内性能。事实上，在体外优化过程中，我们强调了识别多个不同 UBP 的重要性，这些 UBP 的组成核苷酸具有不同的物理化学性质，以便在将它们部署到 SSO 中时提供灵活性。（8， 15）
To extend our SAR data to include the restraints of the in vivo SSO environment, we conducted a screen for pairs of unnatural triphosphates that when added to growth media support high level UBP retention. From an examination of 135 candidate UBPs, we generate new SAR data that differs in several interesting ways from that generated in vitro and, remarkably, at least in some cases demonstrates that replication is more permissive in vivo than in vitro. Most importantly, we discover four new UBPs that are more efficiently retained in vivo than either dNaM-d5SICS or dNaM-dTPT3. One of the constituent nucleobases in each of the new UBPs is dTPT3, suggesting that it represents an at least currently optimal solution, but it is paired with different dNaM analogs. The most promising new UBP is retained in sequences where neither dNaM-d5SICS nor dNaM-dTPT3 is retained even with Cas9, and thus represents the most promising UBP identified to date for use in our continuing efforts to develop an SSO that stably stores increased information.
为了扩展我们的 SAR 数据以包括体内 SSO 环境的限制，我们对非天然三磷酸盐对进行了筛选，当这些三磷酸盐添加到生长培养基中时，这些三磷酸盐支持高水平的 UBP 保留。通过对 135 个候选 UBP 的检查，我们生成了新的 SAR 数据，这些数据在几个有趣的方面与体外生成的数据不同，值得注意的是，至少在某些情况下表明，体内复制比体外复制更宽松。最重要的是，我们发现了四种新的 UBP，它们在体内的保留效率高于 dNaM-d 5SICS 或 dNaM-d TPT3。每个新 UBP 中的组成核碱基之一是 dTPT3，这表明它至少代表了当前的最佳解决方案，但它与不同的 dNaM 类似物配对。最有前途的新 UBP 保留在 dNaM-d 5SICS 和 dNaM-d TPT3 均未保留的序列中，即使使用 Cas9，也代表了迄今为止确定的最有前途的 UBP，可用于我们不断努力开发稳定存储更多信息的 SSO。

Experimental Section 实验部分

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General 常规

All bacteria were cultured in 100 μL of liquid 2×YT media (casein peptone 16 g/L, yeast extract 10 g/L, NaCl 5 g/L) supplemented with potassium phosphate (50 mM, pH 7) in 96-well microwell plates. When noted, antibiotics were used at the following concentrations: chloramphenicol, 5 μg/mL; ampicillin, 100 μg/mL. Cell growth, indicated as OD600, was measured using a PerkinElmer EnVision 2103 Multilabel Reader with a 590/20 nm filter. Unless otherwise stated, molecular biology reagents were purchased from New England Biolabs (Ipswich, MA) and were used according to the manufacturer’s protocols. As necessary, purification of nucleic acids was accomplished by microelution columns (Zymo Research Corp; Irvine, CA). All natural oligonucleotides were purchased from IDT (San Diego, CA), and oligonucleotides containing dNaM were synthesized by Biosearch Technologies (Petaluma, CA) with purification by reverse phase cartridge and were kindly provided by Synthorx (La Jolla, CA). Unnatural nucleotide triphosphates were prepared as previously described (Table S2) and confirmed by MALDI-TOF and UV–vis.
所有细菌均在 100 μL 液体 2×YT 培养基 （酪蛋白蛋白胨 16 g/L、酵母提取物 10 g/L、NaCl 5 g/L） 中培养，并在 96 孔微孔板中补充磷酸钾 （50 mM，pH 7）。注意到后，使用以下浓度的抗生素：氯霉素，5 μg/mL;氨苄青霉素，100 μg/mL。使用带有 590/20 nm 滤光片的 PerkinElmer EnVision 2103 多标记读数仪测量细胞生长，表示为 OD600。除非另有说明，否则分子生物学试剂购自 New England Biolabs（马萨诸塞州伊普斯威奇），并按照制造商的方案使用。必要时，通过微量洗脱柱（Zymo Research Corp;加利福尼亚州尔湾市）。所有天然寡核苷酸均购自 IDT（加利福尼亚州圣地亚哥），含有 dNaM 的寡核苷酸由 Biosearch Technologies（加利福尼亚州佩塔卢马）合成，并通过反相小柱纯化，并由 Synthorx（加利福尼亚州拉霍亚）友情提供。如前所述制备非天然核苷酸三磷酸盐 （表 S2） 并通过 MALDI-TOF 和 UV-vis 确认。

Analysis of UBP Retention
瑞联银行留存率分析

Plasmids containing the dNaM-dTPT3 UBP were prepared and used to transform E. coli strain YZ3 as described previously. (10, 14) Following transformation, the SSO was allowed to recover at 37 °C for 1 h in media containing dNaMTP (125 μM) and dTPT3TP (25 μM). Cells were pelleted by centrifugation, resuspended in fresh media lacking unnatural triphosphates, and then used to inoculate cultures containing different pairs of unnatural triphosphates at the specified concentrations. When the cell density reached an OD600 of ∼0.7, cells were pelleted, and plasmids were recovered and PCR amplified with d5SICSTP and a biotinylated analog of dNaMTP. UBP retention was determined by comparing the intensity of the streptavidin shifted and unshifted bands via PAGE as described previously (10, 14) and in Supporting Information. Plasmids with the dNaM-dTPT3 UBP were used as a starting point for the in vivo replication experiments to eliminate the need to construct separate plasmids for every candidate UBP examined and to eliminate differences in UBP retention during in vitro plasmid construction. We note that this requires each dNaM analog to pair with dTPT3 and each dTPT3 analog to pair with dNaM during the first round of replication, and similar pairing is required for the first round amplification during PCR analysis. It should also be noted that this assay detects bulk retention, and inversion of the pair by, for example, self-pairing, is not excluded.
制备含有 dNaM-d TPT3 UBP 的质粒，并如前所述用于转化大肠杆菌菌株 YZ3。（10， 14）转化后，让 SSO 在含有 dNaMTP （125 μM） 和 dTPT3TP （25 μM） 的培养基中在 37 °C 下恢复 1 小时。通过离心沉淀细胞，重悬于不含非天然三磷酸盐的新鲜培养基中，然后用于接种含有指定浓度的不同非天然三磷酸盐对的培养物。当细胞密度达到 OD600 ∼0.7 时，将细胞沉淀，回收质粒，并用 d5SICSTP 和 dNaMTP 的生物素化类似物进行 PCR 扩增。如前所述 （10， 14） 和支持信息中所述，通过 PAGE 比较链霉亲和素移动和未移动条带的强度来确定 UBP 保留。使用带有 dNaM-d TPT3 UBP 的质粒作为体内复制实验的起点，以消除为检查的每个候选 UBP 构建单独质粒的需要，并消除体外质粒构建过程中 UBP 保留的差异。我们注意到，这需要在第一轮复制期间每个 dNaM 类似物与 dTPT3 配对，每个 dTPT3 类似物与 dNaM 配对，并且在 PCR 分析期间第一轮扩增需要类似的配对。还应该注意的是，该测定检测体积保留，并且不排除通过例如自我配对来反转对。

Results 结果

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To create a template for in vivo replication assays, Golden Gate assembly was used to construct a derivative of the pUC19 plasmid in which a single dNaM-dTPT3 UBP was embedded within the TK1 sequence (local sequence AXT, X = dNaM; referred to hereafter as sequence context 1), a context within which the dNaM-dTPT3 UBP is well replicated in our SSO. (14) Plasmids were then used to transform the SSO, which was allowed to recover briefly in media containing dNaMTP and dTPT3TP. Upon resuspension in fresh media lacking triphosphates, the SSO culture was split into 100 μL aliquots and supplemented with varying concentrations of different pairs of unnatural triphosphates. Once the cultures reached an OD600 of ∼0.7, plasmids were recovered and analyzed for UBP retention (Figure 2).
为了创建用于体内复制测定的模板，使用 Golden Gate 组装构建 pUC19 质粒的衍生物，其中单个 dNaM-d TPT3 UBP 嵌入 TK1 序列（局部序列 AX T，X = dNaM;以下称为序列上下文 1）中，在该上下文中，dNaM-d TPT3 UBP 在我们的 SSO 中很好地复制。（14） 然后使用质粒转化 SSO，使其在含有 dNaMTP 和 dTPT3TP 的培养基中短暂恢复。在缺乏三磷酸盐的新鲜培养基中重悬后，将 SSO 培养物分成 100 μL 等分试样，并补充不同浓度的不同非天然三磷酸盐对。一旦培养物达到 OD600 ∼0.7，回收质粒并分析 UBP 保留（图 2）。

Figure 2 图 2

Figure 2. Graphical representation of screen workflow. For sequence contexts 1–4, X denotes NaM or an NaM analog and Y denotes TPT3 or a TPT3 analog. Retention levels of dNaM-dTPT3 are indicated with “+” symbols. See ref 14 for original report of dNaM-dTPT3 retention data.
图 2.屏幕工作流的图形表示。对于序列上下文 1-4，X 表示 NaM 或 NaM 类似物，Y 表示 TPT3 或 TPT3 类似物。dNaM-d TPT3 的保留水平用“+”符号表示。参见参考文献 14 了解 dNaM-d TPT3 保留数据的原始报告。

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In a first phase of screening, we explored the addition of 25 μM of dTPT3TP and one of 75 different dNaMTP analogs (structures shown in Figure 3A) added at a concentration of 125 μM or 10 μM. After plasmid recovery, we observed UBP retention of >90% with 13 analogs (dMMO2TP, dDMOTP, dNaMTP, dClMOTP, dCNMOTP, d5FMTP, dFDMOTP, dFIMOTP, dZMOTP, dIMOTP, dMIMOTP, dFEMOTP, and dMMO2ATP) (Figure 4A and Supporting Information). Of the remaining analogs, four showed a retention of 50–90% (d2OMeTP, dTfMOTP dMEMOTP, dVMOTP), nine showed a retention of 20–50% (dDM5TP, d2MNTP, d45DMPyTP, dEMOTP, dDMTP, dTOK581TP, dTOK587TP, dPyMO2TP, d35DMPyTP), and the remainder showed a retention of <20%. Addition of the dNaMTP analogs at the lower concentration resulted in generally less efficient UBP retention, with only four, dMMO2TP, dClMOTP, dCNMOTP, and d5FMTP, resulting in high retention (>80%). Five, dFIMOTP, dIMOTP, dFEMOTP, dMMO2ATP, as well as dNaMTP itself, showed intermediate levels of retention (between 40–80%), and four, dFDMOTP, dVMOTP, d2OMeTP, and dZMOTP, showed slightly less retention (20–40%), with the remainder showing <20% retention.
在筛选的第一阶段，我们探索了添加 25 μM 的 dTPT3TP 和 75 种不同的 dNaMTP 类似物中的一种（结构如图 3A），添加浓度为 125 μM 或 10 μM。质粒回收后，我们观察到 13 个类似物（dMMO2TP、dDMOTP、dNaMTP、dClMOTP、dCNMOTP、d5FMTP、dFDMOTP、dFIMOTP、dZMOTP、dIMOTP、dMIMOTP、dFEMOTP 和 dMMO2ATP）的 UBP 保留率为 >90%（图 4A 和支持信息).在其余类似物中，4 个显示保留率为 50-90%（d2OMeTP、dTfMOTP、dMEMOTP、dVMOTP），9 个显示保留率为 20-50%（dDM5TP、d2MNTP、d45DMPyTP、dEMOTP、d DM TP、dTOK581TP、dTOK587TP、dPyMO2TP、d35DMPyTP）， 其余结果显示，较低浓度的 <20 %. Addition of the dNaMTP 类似物的保留导致 UBP 保留效率通常较低，只有四种，dMMO2TP、dClMOTP、dCNMOTP 和 d5FMTP，导致高保留 （>80%）。5 个，dFIMOTP、dIMOTP、dFEMOTP、dMMO2ATP 以及 dNaMTP 本身，显示出中等水平的保留（在 40-80% 之间），4 个，dFDMOTP、dVMOTP、d2OMeTP 和 dZMOTP，显示略低的保留 （20-40%），其余显示 <20% 的保留。

Figure 3 图 3

Figure 3. Structure of analogs used in current study. Shading denotes differences in retention observed in phase 1 of the screen. (A) dNaMTP analogs. Blue shading corresponds to analogs that at 125 μM showed retentions of greater than 90%; green, 50% to 90%; yellow, 20% to 50%; red, <20%. (B) dTPT3TP analogs. Blue shading corresponds to those that at 10 μM showed retentions greater than 90%; red, <10%. Ribose and phosphates omitted for clarity. For original references see Supporting Information.
图 3.当前研究中使用的类似物的结构。阴影表示在屏幕的第 1 阶段观察到的保留差异。（A） dNaMTP 类似物。蓝色阴影对应于在 125 μM 时显示保留率大于 90% 的类似物;绿色，50% 至 90%;黄色，20% 至 50%;红色，<20 %. (B) dTPT3TP 类似物。蓝色阴影对应于 10 μM 时显示保留率大于 90% 的那些;红色，<10 %. Ribose and phosphates omitted for clarity. For original references see 支持信息。

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Figure 4 图 4

Figure 4. UBP retention (%). (A) dNaMTP analogs added at either 125 μM or 10 μM and dTPT3TP added at 25 μM. (B) dTPT3TP analogs added at either 125 μM or 10 μM and dNaMTP added at 125 μM. (C) Selected analogs screened against each other with dNaMTP analogs added at 25 μM and dTPT3TP analogs added at 10 μM. Data are an average of three independent trials, with error bars indicating the standard deviation. A single asterisk indicates that no cell growth was observed at the higher concentration, and a double asterisk indicates that no cell growth was observed at either concentration.
图 4.瑞联银行留存率 （%）。（A） 添加量为 125 μM 或 10 μM 的 dNaMTP 类似物，添加量为 25 μM 的 dTPT3TP。（B） 添加量为 125 μM 或 10 μM 的 dTPT3TP 类似物和添加量为 125 μM 的 dNaMTP。（C） 用 25 μM 的 dNaMTP 类似物和 10 μM 的 dTPT3TP 类似物相互筛选的选定类似物。数据是 3 项独立试验的平均值，误差线表示标准差。单星号表示在较高浓度下未观察到细胞生长，双星号表示在任一浓度下均未观察到细胞生长。

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We next explored UBP retention with the addition of 125 μM dNaMTP and one of 16 different dTPT3TP analogs (structures shown in Figure 3B) at a concentration of 125 μM or 10 μM. When provided at the higher concentration, nine of the dTPT3TP analogs, dTPT3PATP, dTPT3TP, dSICSTP, dFPT1, d4SICS, dTPT1, d5SICS, dNICS, and dSNICS, showed significant UBP retention upon plasmid recovery (Figure 4B and Supporting Information). Unlike with the dNaMTP analogs, these nine UBPs showed similar or better retention when provided at the lower concentration, while dICSTP, d4MICSTP, and d5MICSTP also showed significant retention (retention of these three analogs could not be determined at higher concentrations due to toxicity). Clearly UBP retention is more optimal with the lower concentration of these analogs, and under these conditions, when combined with dNaMTP, all triphosphate analogs examined except dONICSTP, d7OTPTP, d7OFPTP, and d4OTPTP (which were toxic at both concentrations) showed retention of the UBP in excess of 70%.
接下来，我们通过添加 125 μM dNaMTP 和 16 种不同的 dTPT3TP 类似物中的一种（结构如图 3B 所示）来探索 UBP 保留，浓度为 125 μM 或 10 μM。当以较高浓度提供时，dTPT3TP 类似物中的 9 个，dTPT3PATP、dTPT3TP、dSICSTP、dFPT1、d4SICS、dTPT1、d5SICS、dNICS 和 dSNICS，在质粒回收时显示出显著的 UBP 保留（图 4B 和支持信息）。与 dNaMTP 类似物不同，这 9 种 UBP 在较低浓度下表现出相似或更好的保留性，而 dICSTP、d4MICSTP 和 d5MICSTP 也显示出显著的保留性（由于毒性，无法确定这三种类似物在较高浓度下的保留性）。显然，这些类似物的较低浓度下 UBP 保留效果更佳，在这些条件下，当与 dNaMTP 结合使用时，除 dONICSTP、d7OTPTP、d7OFPTP 和 d4OTPTP（在两种浓度下均有毒）外，所有检查的三磷酸类似物均显示 UBP 保留率超过 70%。
In a second phase of screening, we crossed the 12 most promising dTPT3TP analogs with the four most promising dNaMTP analogs identified in the first phase. We incorporated the UBP within the same plasmid, but embedded it within a local sequence of AXA (context 2, X = dNaM), a context in which we have found retention of dNaM-dTPT3 to be more challenging than sequence context 1. (14) Based on the first phase of screening, we also focused on concentrations of the dNaMTP and dTPT3TP analogs of 25 μM and 10 μM, respectively, to increase the dynamic range of the screen. Significant retention was only observed with pairs containing dTPT3TP or dSICSTP, but each was found to yield at least moderate retention with each of the four dNaMTP analogs (Figure 4C and Supporting Information). Retention with dSICSTP was moderate when paired with dMMO2TP (19%), but more significant with d5FMTP, dCNMOTP, and dClMOTP, with 68%, 79%, and 61% retention, respectively. The highest retentions, however, were observed with dTPT3TP (all >87%).
在第二阶段的筛选中，我们将 12 个最有前途的 dTPT3TP 类似物与第一阶段确定的 4 个最有前途的 dNaMTP 类似物杂交。我们将 UBP 掺入同一质粒中，但将其嵌入 AXA 的局部序列中（上下文 2，X = d NaM），在这种情况下，我们发现保留 dNaM-d TPT3 比序列上下文 1 更具挑战性。（14） 基于第一阶段的筛选，我们还关注了 dNaMTP 和 dTPT3TP 类似物的浓度分别为 25 μM 和 10 μM，以增加筛选的动态范围。仅在含有 dTPT3TP 或 dSICSTP 的对中观察到显著的保留，但发现每种对四种 dNaMTP 类似物中的每一种都产生至少中等的保留（图 4C 和支持信息）。与 dMMO2TP 配对时，dSICSTP 的保留率适中 （19%），但 d5FMTP 、 dCNMOTP 和 dClMOTP 的保留率更高，分别为 68% 、 79% 和 61% 的保留率。然而，在 dTPT3TP 中观察到最高的保留率 （均为 >87%）。
Next we explored retention with the four most promising UBPs identified, d5FM-dTPT3, dMMO2-dTPT3, dCNMO-dTPT3, and dClMO-dTPT3, when embedded within context 2, but with unnatural triphosphate concentrations of 25 μM, 10 μM, or 2.5 μM (Figure 5). While it was clear that dMMO2TP and d5FMTP were retained best at 25 μM, UBP retention was too high to differentiate in the case of dCNMOTP and dClMOTP. Thus, we examined retention with the UBP positioned in the same plasmid, but within the local sequence context of CXC (context 3, X = dNaM), which is particularly challenging for dNaM-dTPT3 retention (14) (Figure 5). The data reveal that 25 μM is the most optimal concentration for both pairs and that dCNMOTP and dTPT3TP perform better than dClMOTP and dTPT3TP, with retentions of 42% and 21%, respectively. While d5FMTP and dMMO2TP resulted in slightly higher retention in this sequence context at high concentration (49% and 45%, respectively), they resulted in significantly less retention at the lower concentrations.
接下来，我们探索了鉴定出的四种最有前途的 UBP，d5FM-d TPT3、dMMO2-d TPT3、dCNMO-d TPT3 和 dClMO-d TPT3，当嵌入上下文 2 中时，但三磷酸盐浓度为 25 μM、10 μM 或 2.5 μM（图 5）。虽然很明显 dMMO2TP 和 d5FMTP 在 25 μM 时保留效果最好，但 UBP 保留率太高，无法区分 dCNMOTP 和 dClMOTP。因此，我们检查了位于同一质粒中的 UBP 的保留，但在 CXC 的局部序列上下文中（上下文 3，X = d NaM），这对于 dNaM-d TPT3 保留尤其具有挑战性 （14）（图 5）。数据显示，25 μM 是两对的最佳浓度，dCNMOTP 和 dTPT3TP 的性能优于 dClMOTP 和 dTPT3TP，保留率分别为 42% 和 21%。虽然 d5FMTP 和 dMMO2TP 在该序列背景下在高浓度下导致略高的保留 （分别为 49% 和 45%），但它们在较低浓度下的保留率显着降低。

Figure 5 图 5

Figure 5. UBP retention (%) with dTPT3TP and different dNaMTP analogs added at varying concentrations to the media. Shading indicates the level of UBP retention. Values are the average and standard deviation of three independent determinations.
图 5.向培养基中加入不同浓度的 dTPT3TP 和不同的 dNaMTP 类似物时的 UBP 保留率 （%）。底纹表示 UBP 保留级别。值是三个独立测定的平均值和标准差。

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Of the 135 candidate UBPs examined, the data reveal that dCNMO-dTPT3 is most efficiently replicated in the SSO. To directly and more thoroughly compare this UBP with dNaM-dTPT3, the most efficiently replicated UBP previously identified, we examined retention of both pairs in the three sequence contexts described above, as well as a fourth, which positions the UBP within the local sequence context of CXG (context 4), which is one of the most challenging sequences for dNaM-dTPT3 (14) (Figure 6). dTPT3TP was added at a fixed concentration of 25 μM, while dNaMTP or dCNMOTP was added at a concentration of either 125 μM or 25 μM. At the higher concentration, we observed >99% retention in sequence context 1 with both dCNMOTP and dNaMTP, but while retention remained high with dCNMOTP added at the lower concentration (98%), it was decreased with dNaMTP (85%). In context 2, reduced retention was observed with dNaMTP at both the high concentration (73%) and the low concentration (36%), but retention at both concentrations remained high with dCNMOTP (>99%). In context 3, addition of dNaMTP at the higher concentration resulted in only moderate retention (26%), while addition at the lower concentration resulted in no retention. However, with dCNMOTP, significant retention was observed at both high (65%) and low concentrations (42%). Finally, with context 4, the UBP was not retained significantly at either concentration with dNaMTP, but remained moderate at high concentrations of dCNMOTP (24%).
在检查的 135 个候选 UBP 中，数据显示 dCNMO-d TPT3 在 SSO 中的复制效率最高。为了直接、更彻底地将该 UBP 与先前确定的复制效率最高的 UBP dNaM-d TPT3 进行比较，我们检查了上述三个序列上下文中两个对的保留情况，以及第四个序列，它将 UBP 定位在 CXG 的局部序列上下文中（上下文 4），这是 dNaM-d TPT3 最具挑战性的序列之一 （14）（图 6）。dTPT3TP 的添加浓度为 25 μM，而 dNaMTP 或 dCNMOTP 的添加浓度为 125 μM 或 25 μM。在较高浓度下，我们在序列上下文 1 中观察到 dCNMOTP 和 dNaMTP 的保留率为 >99%，但是虽然在较低浓度 （98%） 下添加 dCNMOTP 的保留率仍然很高，但随着 dNaMTP （85%） 的添加而降低。在上下文 2 中，在高浓度 （73%） 和低浓度 （36%） 下观察到 dNaMTP 的保留率降低，但使用 dCNMOTP （>99%） 在两种浓度下的保留率仍然很高。在上下文 3 中，添加较高浓度的 d NaM TP 仅导致中等保留 （26%），而添加较低浓度的 dNaMTP 导致没有保留。然而，对于 dCNMOTP，在高浓度 （65%） 和低浓度 （42%） 下均观察到显着保留。 最后，在上下文 4 中，UBP 在任一浓度下均未显著保留 dNaMTP，但在高浓度d CNMOTP （24%） 下保持适中。

Figure 6 图 6

Figure 6. UBP retention (%) with 25 μM dTPT3TP and varying concentrations of dNaMTP or dCNMOTP added to the media. Shading indicates the level of UBP retention. Values are the average and standard deviation of three independent determinations.
图 6.在培养基中添加 25 μM dTPT3TP 和不同浓度的 dNaMTP 或 dCNMOTP 后，UBP 保留率 （%）。底纹表示 UBP 保留级别。值是三个独立测定的平均值和标准差。

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Discussion 讨论

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The discovery of dNaM-dTPT3 was driven by in vitro SARs that ultimately drew on over 150 unnatural nucleotides. While dNaM-dTPT3 was the most promising UBP discovered and is clearly suitable for use within a living SSO, (14) its retention is sequence context-dependent, with some sequences showing high retention and others less or none. During the in vitro discovery phase, we also identified variants whose constituent nucleotides have distinct physicochemical properties that may differentiate performance in vivo. With these analogs, we have now examined 135 variant UBPs within the in vivo environment of our SSO. Interestingly, we find in vivo SARs that are both similar to and different from those collected in vitro. For example, dICS and its methyl-derivatized analogs d4MICS and d5MICS support UBP retention in vivo reasonably well, but only at low concentration, suggesting that they are misincorporated opposite natural nucleotides at high concentrations, thereby resulting in stalled replication forks and toxicity. Indeed, in vitro, steady-state kinetic analyses suggest that these analogs can be misinserted opposite natural nucleotides in a template with reasonable efficiency. (16) However, UBPs containing these analogs cannot be PCR amplified, suggesting that the observed retention is unique to the in vivo environment. Heteroatom derivatization of the dICS scaffold is generally deleterious and results in significant toxicity, again consistent with misincorporation, as we observed previously in vitro. (17) An exception is dNICS, as this heteroatom-derivatized analog of dICS supports UBP retention reasonably well, and in fact, the additional sulfur substituent of dSNICS results in an analog that supports moderate retention at both low and high concentrations. The beneficial effect of the sulfur does not depend on aza substitution, as retention is also increased with dSICS compared to dICS. Thus, the aza and sulfur substituents appear to independently reduce mispairing in vivo. While this was observed in vitro for the sulfur substituent, the opposite was observed with aza substitution, suggesting that its ability to reduce mispairing is unique to the in vivo environment.
dNaM-d TPT3 的发现是由体外 SAR 驱动的，该 SAR 最终利用了 150 多个非天然核苷酸。虽然 dNaM-d TPT3 是发现的最有前途的 UBP，并且显然适合在活体 SSO 中使用，（14） 但它的保留性与序列上下文有关，一些序列显示出高保留率，而另一些序列则较少或没有保留。在体外发现阶段，我们还鉴定了其组成核苷酸具有不同物理化学性质的变体，这些变体可能会区分体内性能。通过这些类似物，我们现在已经在 SSO 的体内环境中研究了 135 个变体 UBP。有趣的是，我们发现体内 SAR 与体外收集的 SAR 既相似又不同。例如，dICS 及其甲基衍生类似物 d4MICS 和 d5MICS 相当好地支持 UBP 在体内的保留，但仅限于低浓度，这表明它们在高浓度下被错误掺入相反的天然核苷酸，从而导致复制叉停滞和毒性。事实上，在体外，稳态动力学分析表明，这些类似物可以以合理的效率错误地插入模板中天然核苷酸的相反位置。（16） 然而，含有这些类似物的 UBP 不能进行 PCR 扩增，这表明观察到的保留是体内环境所特有的。dICS 支架的杂原子衍生化通常是有害的，并导致显着的毒性，正如我们之前在体外观察到的那样，这与错误掺入一致。 （17） dNICS 是一个例外，因为这种杂原子衍生的 dICS 类似物相当好地支持 UBP 保留，事实上，dSNICS 的额外硫取代基导致类似物在低浓度和高浓度下都支持中等保留。硫的有益作用不依赖于 aza 取代，因为与 dICS 相比，dSICS 的保留率也有所增加。因此，aza 和硫取代基似乎可以独立地减少体内错配。虽然在体外观察到硫取代基的这种情况，但在 aza 取代中观察到相反的情况，这表明其减少错配的能力是体内环境独有的。
The modification of unnatural nucleotides with linkers that allow for site-specific attachment of different functionalities is of particular interest for in vivo labeling experiments. Linker modification of dTPT3TP, resulting in dTPT3PATP, is well tolerated in vivo, as was also observed in vitro. However, dMMO2ATP is reasonably well tolerated in vivo, while dMMO2PATP, dMMO2BIOTP, and dMMO2SSBIOTP completely ablate retention, contrary to what is observed in vitro. (18) While dMMO2ATP shows a decrease in retention at low concentration, which dTPT3PATP does not, its free amine linker should facilitate in vivo labeling or cross-linking experiments. Similarly, dZMOTP and dFEMOTP are well retained in vivo when supplemented at high concentrations and provide an azide and alkyne moiety in the major groove, respectively, where they should also facilitate in vivo labeling or cross-linking.
使用允许不同功能的位点特异性连接的接头修饰非天然核苷酸对于体内标记实验特别感兴趣。dTPT3TP 的接头修饰，导致 dTPT3PATP，在体内耐受性良好，在体外也观察到。然而，dMMO2ATP 在体内具有良好的耐受性，而 dMMO2PATP、dMMO2BIOTP 和 dMMO2SSBIOTP 完全消融保留，这与在体外观察到的情况相反。（18） 虽然 dMMO2ATP 在低浓度下显示保留性降低，而 dTPT3PATP 则没有，但其游离胺接头应有助于体内标记或交联实验。同样，dZMOTP 和 dFEMOTP 在高浓度补充时在体内保留良好，并分别在大沟中提供叠氮化物和炔烃部分，它们也应促进体内标记或交联。
A large body of in vitro SAR data demonstrates convincingly that an H-bond acceptor positioned ortho to the glycosidic bond, and thus oriented into the developing minor groove upon incorporation into DNA, is generally required for efficient continued primer elongation. (19) The general requirement of an H-bond acceptor at this position is consistent with studies of natural base pairs, (20-23) which invariably have a similarly disposed H-bond acceptor that is thought to engage in critical interactions with polymerase-based H-bond donors. (24) An exception is the relatively efficient PCR amplification of DNA containing d2MN paired opposite dTPT3. (8)In vivo, d2MNTP also supports retention with dTPT3TP, but when combined with dTPT3TP, dDM5TP does as well. Moreover, while dICSTP, dNICSTP, d4MICSTP, and d5MICSTP do not support PCR amplification when paired with any analog, (8) they support reasonable retention in vivo when paired dNaMTP. Clearly, the requirements for the ortho group are somewhat different in vitro and in vivo, and at least in some cases, they are more permissive in vivo.
大量体外 SAR 数据令人信服地表明，通常需要一个 H 键受体与糖苷键邻位，从而在掺入 DNA 后定向到发育中的小沟中，这是有效持续引物延伸所必需的。（19） 该位置对 H 键受体的一般要求与天然碱基对的研究一致，（20-23） 它们总是具有一个配置相似的 H 键受体，该受体被认为与基于聚合酶的 H 键供体发生关键相互作用。（24） 一个例外是含有 d2MN 与 dTPT3 对对的 DNA 的相对有效的 PCR 扩增。（8）在体内，d2MNTP 也支持 dTPT3TP 的保留，但当与 dTPT3TP 结合时，dDM5TP 也支持保留。此外，虽然 dICSTP、dNICSTP、d4MICSTP 和 d5MICSTP 在与任何类似物配对时不支持 PCR 扩增，（8） 当配对 dNaMTP 时，它们支持合理的体内保留。显然，对邻位基团的要求在体外和体内略有不同，至少在某些情况下， 它们在体内更宽松。
From a practical perspective, the most important results of the current study are the excellent in vivo performance of the d5FM-dTPT3, dMMO2-dTPT3, dCNMO-dTPT3, and dClMO-dTPT3 UBPs (Figure 7). Retention of each of these new UBPs in the SSO, in particular, dCNMO-dTPT3, is better than that of dNaM-dTPT3, which previously was the most promising UBP identified, and requires the addition of less nucleotide triphosphate to the growth media. In fact, dCNMO-dTPT3 shows at least moderate retention in sequence context 4, where dNaM-dTPT3 is retained so poorly that it cannot be rescued by Cas9, suggesting that it is lost immediately upon attempted replication. Interestingly, this contrasts with in vitro data where retention of dNaM-dTPT3 is better than retention of dCNMO-dTPT3, (8, 15) suggesting that E. coli provides a unique environment for which dCNMO-dTPT3 is more optimal. Possible contributing factors include PtNTT2-mediated uptake, stability within the cell, or recognition by different polymerases that can access the replication fork and actually mediate replication in vivo.
从实践的角度来看，当前研究最重要的结果是 d5FM-d TPT3、dMMO2-d TPT3、dCNMO-d TPT3 和 dClMO-d TPT3 UBP 的优异体内性能（图 7）。这些新 UBP 中的每一个在 SSO 中的保留率，特别是 dCNMO-d TPT3，都优于 dNaM-d TPT3，后者是以前确定的最有前途的 UBP，并且需要向生长培养基中添加较少的核苷酸三磷酸。事实上，dCNMO-d TPT3 在序列上下文 4 中显示出至少中等的保留，其中 dNaM-d TPT3 保留得非常差，以至于无法被 Cas9 挽救，这表明它在尝试复制后立即丢失。有趣的是，这与体外数据形成鲜明对比，其中 dNaM-d TPT3 的保留优于 dCNMO-d TPT3 的保留，（8， 15） 表明大肠杆菌提供了一个独特的环境，其中 dCNMO-d TPT3 更优化。可能的促成因素包括 PtNTT2 介导的摄取、细胞内的稳定性或不同聚合酶的识别，这些聚合酶可以访问复制叉并实际介导体内复制。

Figure 7 图 7

Figure 7. Four optimal UBPs discovered in this work.
图 7.本研究中发现的四个最优 UBP。

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Regardless of the specific properties that underlie their performance, it is clear that the present work has identified four new UBPs that now represent the most promising candidates for use in an SSO. These new UBPs further demonstrate the ability of hydrophobic and packing interactions to replace complementary H-bonding as the force underlying information storage. It is interesting that each of the new dNaM analogs bear smaller, single ring nucleobases. While this may have contributed to their in vivo performance by facilitating uptake, they are likely to be less prone to cross-strand intercalation and more likely to adopt edge-to-edge structures. This may also contribute to their more optimal retention and possibly even facilitate the replication of DNA with higher density UBPs. Only UBP loss was characterized in this study, as mutations involving natural nucleotides are predicted to be most problematic. (5, 25, 26) However, cross-strand intercalation may also facilitate self-pairing, which even at low levels could cause UBP inversion (where the individual nucleotides switch strands). Whatever its level, self-pairing mediated UBP inversion may be less likely with these analogs. Finally, the performance of each of the new UBPs is likely to be even further improved by use of Cas9, and we are currently exploring this possibility. The availability of a family of UBPs that are well retained in the in vivo environment of the SSO, but that also possess distinct physicochemical properties, is of great significance as our efforts to retrieve the increased information via transcription and translation will likely introduce additional requirements and restraints.
无论其性能背后的具体属性如何，很明显，本研究已经确定了四个新的 UBP，它们现在代表了在 SSO 中使用的最有希望的候选者。这些新的 UBP 进一步证明了疏水和堆积相互作用取代互补 H 键作为信息存储基础的力量的能力。有趣的是，每个新的 dNaM 类似物都带有更小的单环核碱基。虽然这可能通过促进摄取来促进它们的体内性能，但它们可能不太容易出现交叉链插层，并且更有可能采用边缘到边缘的结构。这也可能有助于它们更理想的保留，甚至可能促进具有更高密度 UBP 的 DNA 复制。本研究仅表征了 UBP 丢失，因为预计涉及天然核苷酸的突变是最有问题的。（5、25、26）然而，交叉链嵌入也可能促进自配对，即使在低水平下也可能导致 UBP 倒置（单个核苷酸交换链）。无论其水平如何，这些类似物的自配对介导的 UBP 倒置的可能性都较小。最后，使用 Cas9 可能会进一步提高每个新 UBP 的性能，我们目前正在探索这种可能性。在 SSO 的体内环境中保留良好但同时也具有不同物理化学特性的 UBP 家族的可用性具有重要意义，因为我们通过转录和翻译检索增加的信息的努力可能会引入额外的要求和限制。

Supporting Information 支持信息

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.7b03540.
支持信息可在 ACS Publications 网站免费获取，网址为 DOI：10.1021/jacs.7b03540。

  • Additional methods, supporting tables and figures, and references (PDF)
    其他方法、支持表格和图表以及参考资料 （PDF）

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Author Information 作者信息

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  • Corresponding Author 通讯作者
  • Author 作者
    • Aaron W. Feldman - Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, United States
      Aaron W. Feldman - 斯克里普斯研究所化学系，10550 North Torrey Pines Road， La Jolla， California 92037， 美国
  • Notes 笔记
    The authors declare no competing financial interest.
    作者声明没有竞争性的经济利益。

Acknowledgment 确认

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This work was supported by the National Institutes of Health (grant nos. GM060005 and GM118178 to F.E.R.). A.W.F. was supported by a National Science Foundation Graduate Research Fellowship (grant no. NSF/DGE-1346837).
这项工作得到了美国国立卫生研究院 （grant nos.GM060005 和 F.E.R. GM118178）。AWF 得到了美国国家科学基金会研究生研究奖学金（资助号。NSF/DGE-1346837）。

References 引用

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This article references 26 other publications.
本文引用了其他 26 种出版物。

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    A review. All biol. information, since the last common ancestor of all life on Earth, has been encoded by a genetic alphabet consisting of only four nucleotides that form two base pairs. Long-standing efforts to develop two synthetic nucleotides that form a third, unnatural base pair (UBP) have recently yielded three promising candidates, one based on alternative hydrogen bonding, and two based on hydrophobic and packing forces. All three of these UBPs are replicated and transcribed with remarkable efficiency and fidelity, and the latter two thus demonstrate that hydrogen bonding is not unique in its ability to underlie the storage and retrieval of genetic information. This Review highlights these recent developments as well as the applications enabled by the UBPs, including the expansion of the evolution process to include new functionality and the creation of semi-synthetic life that stores increased information.
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    Angewandte Chemie，国际版 （2015 年）， 54 （41）、 公元 11930 年 - 11944 元科登： ACIEF5; 国际标准书号：1433-7851。 （Wiley-VCH Verlag GmbH & Co. KGaA）
    评论。 自地球上所有生命的最后一个共同祖先以来，所有生物信息都是由一个遗传字母编码的，该字母表仅由四个核苷酸组成，形成两个碱基对。 长期以来，人们一直在努力开发形成第三个非天然碱基对 （UBP） 的合成核苷酸，最近产生了三种有前途的候选核苷酸，一种基于替代氢键，另一种基于疏水和堆积力。 所有这三种 UBP 都以极高的效率和保真度进行复制和转录，因此，后两者表明氢键在遗传信息的存储和检索能力方面并不独特。 本综述重点介绍了这些最新发展以及 UBP 实现的应用，包括扩展进化过程以包括新功能和创建存储更多信息的半合成生命。
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    Zhang, L.; Yang, Z.; Sefah, K.; Bradley, K. M.; Hoshika, S.; Kim, M. J.; Kim, H. J.; Zhu, G.; Jimenez, E.; Cansiz, S.; Teng, I. T.; Champanhac, C.; McLendon, C.; Liu, C.; Zhang, W.; Gerloff, D. L.; Huang, Z.; Tan, W.; Benner, S. A. J. Am. Chem. Soc. 2015, 137, 6734– 6737 DOI: 10.1021/jacs.5b02251
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    Axiomatically, the d. of information stored in DNA, with just four nucleotides (GACT), is higher than in a binary code, but less than it might be if synthetic biologists succeed in adding independently replicating nucleotides to genetic systems. Such addn. could also add functional groups not found in natural DNA, but useful for mol. performance. Here, we consider two new nucleotides (Z and P, 6-amino-5-nitro-3-(1'-β-D-2'-deoxyribo-furanosyl)-2(1H)-pyridone and 2-amino-8-(1'-β-D-2'-deoxyribofuranosyl)-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one). These are designed to pair via complete Watson-Crick geometry. These were added to a library of oligonucleotides used in a lab. in vitro evolution (LIVE) expt.; the GACTZP library was challenged to deliver mols. that bind selectively to liver cancer cells, but not to untransformed liver cells. Unlike in classical in vitro selection, low levels of mutation allow this system to evolve to create binding mols. not necessarily present in the original library. Over a dozen binding species were recovered. The best had Z and/or P in their sequences. Several had multiple, nearby, and adjacent Zs and Ps. Only the weaker binders contained no Z or P at all. This suggests that this system explored much of the sequence space available to this genetic system and that GACTZP libraries are richer reservoirs of functionality than std. libraries.
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    张 L.; 杨 Z.; 塞法 K.; 布拉德利，KM; 星香 S.; 金 M.J.; 金 （Kim， H.J.）; 朱 G.; 希门尼斯，E.; 坎西兹，S.; 滕，IT。; 尚帕尼亚克; 麦克伦登 C.; 刘 C.; 张伟; Gerloff， D. L.; 黄 Z.; 时间，W.; 本纳，SAJ. Am. Chem. Soc. 2015， 137， 6734–6737 DOI： 10.1021/jacs.5b02251
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    功能性六核苷酸DNA的进化
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    从公理上讲，存储在 DNA 中的信息 d. 只有四个核苷酸 （GACT），高于二进制代码中的d.，但低于合成生物学家成功地将独立复制的核苷酸添加到遗传系统时可能达到的水平。 这种添加还可以添加天然 DNA 中没有的官能团，但对分子性能很有用。 在这里，我们考虑了两个新的核苷酸（Z 和 P，6-氨基-5-硝基-3-（1'-β-D-2'-脱氧核糖基-呋喃糖基）-2（1H）-吡啶酮和 2-氨基-8-（1'-β-D-2'-脱氧呋喃核糖基）-咪唑[1,2-a]-1,3,5-三嗪-4（8H）-酮）。 这些旨在通过完整的 Watson-Crick 几何结构进行配对。 这些被添加到实验室使用的寡核苷酸文库中。体外进化 （LIVE） 实验;GACTZP 文库面临交付 mols 的挑战。选择性地与肝癌细胞结合，但不与未转化的肝细胞结合。 与经典的体外选择不同，低水平突变允许该系统进化以产生结合分子。不一定存在于原始库中。 回收了十几种结合物种。 最好的序列中有 Z 和/或 P。 几个项目有多个、附近和相邻的 Z 和 P。 只有较弱的粘合剂根本不包含 Z 或 P。 这表明该系统探索了该遗传系统可用的大部分序列空间，并且 GACTZP 文库是比标准文库更丰富的功能库。
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    Kimoto, M.; Hirao, I. In Chemical Biology of Nucleic Acids: Fundamentals and Clinical Applications; Erdmann, A. V.; Markiewicz, T. W.; Barciszewski, J., Eds.; Springer: Berlin, Heidelberg, 2014; p 131– 148.
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    Lavergne, T.; Malyshev, D. A.; Romesberg, F. E. Chem. - Eur. J. 2012, 18, 1231– 1239 DOI: 10.1002/chem.201102066
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    Major groove substituents and polymerase recognition of a class of predominantly hydrophobic unnatural base pairs
    Lavergne, Thomas; Malyshev, Denis A.; Romesberg, Floyd E.
    Chemistry - A European Journal (2012), 18 (4), 1231-1239, S1231/1-S1231/27CODEN: CEUJED; ISSN:0947-6539. (Wiley-VCH Verlag GmbH & Co. KGaA)
    Expansion of the genetic alphabet with an unnatural base pair is a long-standing goal of synthetic biol. The authors have developed a class of unnatural base pairs, formed between d5SICS and analogs of dMMO2 that are efficiently and selectively replicated by the Klenow fragment (Kf) DNA polymerase. In an effort to further characterize and optimize replication, the authors report the synthesis of five new dMMO2 analogs bearing different substituents designed to be oriented into the developing major groove and an anal. of their insertion opposite d5SICS by Kf and Thermus aquaticus DNA polymerase I (Taq). We also expand the anal. of the previously optimized pair, dNaM-d5SICS, to include replication by Taq. Finally, the efficiency and fidelity of PCR amplification of the base pairs by Taq or Deep Vent polymerases was examd. The resulting structure-activity relationship data suggest that the major determinants of efficient replication are the minimization of desolvation effects and the introduction of favorable hydrophobic packing, and that Taq is more sensitive than Kf to structural changes. In addn., the authors identify an analog (dNMO1) that is a better partner for d5SICS than any of the previously identified dMMO2 analogs with the exception of dNaM. They also found that dNaM-d5SICS is replicated by both Kf and Taq with rates approaching those of a natural base pair.
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    拉弗涅，T.; 马雷舍夫，DA; Romesberg， F. E.化学 - Eur. J. 2012， 18， 1231– 1239 DOI： 10.1002/化学201102066
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    一类主要疏水性非天然碱基对的主要沟取代基和聚合酶识别
    Lavergne， Thomas;马雷舍夫，丹尼斯 A.;罗梅斯伯格，弗洛伊德 E.
    化学 - A European Journal （2012 年）、 18 （4）、 1231-1239、S1231/1-S1231/27科登： CEUJED; 国际标准书号：0947-6539。 （Wiley-VCH Verlag GmbH & Co. KGaA）
    用非自然碱基对扩展遗传字母表是合成生物学的一个长期目标。 作者开发了一类非天然碱基对，形成于 d5SICS 和 dMMO2 类似物之间，它们被 Klenow 片段 （Kf） DNA 聚合酶有效且选择性地复制。 为了进一步表征和优化复制，作者报告了五种带有不同取代基的新型 dMMO2 类似物的合成，这些取代基被设计成通过 Kf 和水生热藻 DNA 聚合酶 I （Taq） 插入与 d5SICS 相反的 d5SICS 的肛门。 我们还扩展了先前优化的对 dNaM-d5SICS 的分析，以包括 Taq 的复制。 最后，检查了 Taq 或 Deep Vent 聚合酶对碱基对的 PCR 扩增的效率和保真度。 由此产生的结构-活性关系数据表明，有效复制的主要决定因素是脱溶剂效应的最小化和有利疏水堆积的引入，并且 Taq 比 Kf 对结构变化更敏感。 在附录中，作者确定了一种类似物 （dNMO1），它比以前确定的任何 dMMO2 类似物都更适合 d5SICS 的伴侣，但 dNaM 除外。 他们还发现 dNaM-d5SICS 可以被 Kf 和 Taq 复制，其速率接近天然碱基对的速率。
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    Malyshev, D. A.; Dhami, K.; Quach, H. T.; Lavergne, T.; Ordoukhanian, P.; Torkamani, A.; Romesberg, F. E. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 12005– 12010 DOI: 10.1073/pnas.1205176109
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    马雷舍夫，DA。; 达米，K.; 夸奇，HT; 拉弗涅，T.; Ordoukhanian， P.; 托卡马尼，A.; Romesberg， F. E.美国国家科学院院刊 2012， 109， 12005– 12010 DOI： 10.1073/pnas.1205176109
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    Dhami, K.; Malyshev, D. A.; Ordoukhanian, P.; Kubelka, T.; Hocek, M.; Romesberg, F. E. Nucleic Acids Res. 2014, 42, 10235– 10244 DOI: 10.1093/nar/gku715
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    达米，K.; 马雷舍夫，DA; Ordoukhanian， P.; 库贝尔卡，T.; 霍切克 M.; Romesberg， F. E.核酸研究 2014， 42， 10235– 10244 DOI： 10.1093/NAR/GKU715
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    Li, L.; Degardin, M.; Lavergne, T.; Malyshev, D. A.; Dhami, K.; Ordoukhanian, P.; Romesberg, F. E. J. Am. Chem. Soc. 2014, 136, 826– 829 DOI: 10.1021/ja408814g
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    李 L.; 德加丁; 拉弗涅，T.; 马雷舍夫，DA; 达米，K.; Ordoukhanian， P.; Romesberg， F. E.J. Am. Chem. Soc. 2014， 136， 826– 829 DOI： 10.1021/JA408814G
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    Malyshev, D. A.; Dhami, K.; Lavergne, T.; Chen, T.; Dai, N.; Foster, J. M.; Correa, I. R., Jr.; Romesberg, F. E. Nature 2014, 509, 385– 388 DOI: 10.1038/nature13314
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    A semi-synthetic organism with an expanded genetic alphabet
    Malyshev, 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.
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    10
    马雷舍夫，DA; 达米，K.; 拉弗涅，T.; 陈 T.; 戴 N.; 福斯特，JM; 小科雷亚; Romesberg， F. E.自然 2014， 509， 385– 388 DOI： 10.1038/自然13314
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    一种具有扩展遗传字母表的半合成生物
    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。
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    Betz, K.; Malyshev, D. A.; Lavergne, T.; Welte, W.; Diederichs, K.; Dwyer, T. J.; Ordoukhanian, P.; Romesberg, F. E.; Marx, A. Nat. Chem. Biol. 2012, 8, 612– 614 DOI: 10.1038/nchembio.966
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    KlenTaq polymerase replicates unnatural base pairs by inducing a Watson-Crick geometry
    Betz, Karin; Malyshev, Denis A.; Lavergne, Thomas; Welte, Wolfram; Diederichs, Kay; Dwyer, Tammy J.; Ordoukhanian, Phillip; Romesberg, Floyd E.; Marx, Andreas
    Nature 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.
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    11
    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.; Romesberg， F. E.; 马克思，A.Nat. Chem. Biol. 2012， 8， 612– 614 DOI： 10.1038/nchembio.966
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    KlenTaq 聚合酶通过诱导 Watson-Crick 几何
    Betz， Karin 复制非自然碱基对;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， Andreas
    Nature 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。
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    Betz, K.; Malyshev, D. A.; Lavergne, T.; Welte, W.; Diederichs, K.; Romesberg, F. E.; Marx, A. J. Am. Chem. Soc. 2013, 135, 18637– 18643 DOI: 10.1021/ja409609j
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    12
    贝茨，K.; 马雷舍夫，DA; 拉弗涅，T.; 韦尔特，W.; 迪德里希斯，K.; Romesberg， F. E.; 马克思，A.J. Am. Chem. Soc. 2013， 135， 18637– 18643 DOI： 10.1021/JA409609J
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    Malyshev, D. A.; Pfaff, D. A.; Ippoliti, S. I.; Hwang, G. T.; Dwyer, T. J.; Romesberg, F. E. Chem. - Eur. J. 2010, 16, 12650– 12659 DOI: 10.1002/chem.201000959
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    13
    Solution Structure, Mechanism of Replication, and Optimization of an Unnatural Base Pair
    Malyshev, 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.
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    13
    马雷舍夫，D. A.; 普法夫，DA; 伊波利蒂，SI; 黄 GT; 德怀尔，TJ; Romesberg， F. E.化学 - Eur. J. 2010， 16， 12650– 12659 DOI： 10.1002/化学201000959
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    13
    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。
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    Protein Science (2001), 10 (6), 1225-1233CODEN: PRCIEI; ISSN:0961-8368. (Cold Spring Harbor Laboratory Press)
    The mechanism by which DNA polymerase I enzymes function has been the subject of extensive biochem. and structural studies. The authors previously detd. the structure of a ternary complex of the large fragment of DNA polymerase I from Thermus aquaticus (Klentaq1) bound to a primer/template DNA and a dideoxycytidine 5'-triphosphate (ddCTP). In this report, the authors present the details of the 2.3-Å resoln. crystal structures of three addnl. ternary complexes of Klentaq1 bound to a primer/template DNA and a dideoxyguanosine 5'-triphosphate (ddGTP), a dideoxythymidine 5'-triphosphate (ddTTP), or a dideoxyadenosine 5'-triphosphate (ddATP). Comparison of the active site of the four ternary complexes reveals that the protein residues around the nascent base pair (that formed between the incoming dideoxynucleoside triphosphate [ddNTP] and the template base) form a snug binding pocket into which only a correct Watson-Crick base pair can fit. Except in the ternary complex bound to dideoxyguanosine 5'-triphosphate, there are no sequence specific contacts between the protein side chains and the nascent base pair, suggesting that steric constraints imposed by the protein onto the nascent base pair is the major contributor to nucleotide selectivity at the polymerase active site. The protein around the polymerase active site also shows plasticity, which may be responsible for the substrate diversity of the enzyme. Two conserved side chains, Q754 and R573, form hydrogen bonds with the N3 atom in the purine base and O2 atom in the pyrimidine base at the minor groove side of the base pair formed by the incorporated ddNMP and the corresponding template base in all the four ternary complexes. These hydrogen-bonding interactions may provide a means of detecting misincorporation at this position.
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    24
    李英; Waksman， G. 蛋白质科学。 2001， 10， 1225–1233 DOI： 10.1110/ps.250101
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    Klentaq1 的 ddATP-、ddTTP-、ddCTP- 和 ddGTP 捕获的三元复合物的晶体结构：核苷酸掺入和选择性的见解
    Li， Ying;Waksman， Gabriel
    蛋白质科学 （2001 年） 10 （6）、 1225 年至 1233 年科登： 普雷西; 国际标准书号：0961-8368。 （冷泉港实验室出版社）
    DNA 聚合酶 I 酶的功能机制一直是广泛的生物化学和结构研究的主题。 作者之前 detd.来自水生热菌 （Klentaq1） 的 DNA 聚合酶 I 大片段的三元复合物的结构，与引物/模板 DNA 和二脱氧胞苷 5'-三磷酸 （ddCTP） 结合。 在本报告中，作者介绍了 2.3-Å 溶液的详细信息。三个 addnl 的晶体结构。Klentaq1 的三元复合物与引物/模板 DNA 和二脱氧鸟苷 5'-三磷酸 （ddGTP）、二脱氧胸苷 5'-三磷酸 （ddTTP） 或 5'-三磷酸二脱氧腺苷 （ddATP） 结合。 四种三元复合物的活性位点的比较表明，新生碱基对周围的蛋白质残基（在进入的二脱氧核苷三磷酸 [ddNTP] 和模板碱基之间形成）形成一个紧密的结合口袋，只有正确的 Watson-Crick 碱基对才能放入其中。 除了与二脱氧鸟苷 5'-三磷酸结合的三元复合物外，蛋白质侧链和新生碱基对之间没有序列特异性接触，这表明蛋白质施加在新生碱基对上的空间限制是聚合酶活性位点核苷酸选择性的主要贡献者。 聚合酶活性位点周围的蛋白质也显示出可塑性，这可能是酶底物多样性的原因。 两条保守的侧链 Q754 和 R573 与嘌呤碱基中的 N3 原子和嘧啶碱基中的 O2 原子形成氢键，位于由掺入的 ddNMP 和所有四个三元配合物中的相应模板碱基形成的碱基对的小沟侧。 这些氢键相互作用可能提供了一种检测该位置错误掺入的方法。
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    Seo, Y. J.; Hwang, G. T.; Ordoukhanian, P.; Romesberg, F. E. J. Am. Chem. Soc. 2009, 131, 3246– 3252 DOI: 10.1021/ja807853m
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    25
    徐，YJ; 黄 GT; Ordoukhanian， P.; Romesberg， F. E.J. Am. Chem. Soc. 2009， 131， 3246– 3252 DOI： 10.1021/JA807853M
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    Morris, S. E.; Feldman, A. W.; Romesberg, F. E. ACS Synth. Biol. 2017, published online June 27. DOI:  DOI: 10.1021/acssynbio.7b00115 .
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    26莫里斯，SE;费尔德曼，AW;Romesberg， FE ACS 合成器。Biol.2017,6 月 27 日在线发布。DOI： DOI： 10.1021/acssynbio.7b00115 .

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  7. Giacomo Padroni, Jamie M. Withers, Andrea Taladriz-Sender, Linus F. Reichenbach, John A. Parkinson, Glenn A. Burley. Sequence-Selective Minor Groove Recognition of a DNA Duplex Containing Synthetic Genetic Components. Journal of the American Chemical Society 2019, 141 (24) , 9555-9563. https://doi.org/10.1021/jacs.8b12444
  8. Vivian T. Dien, Matthew Holcomb, Aaron W. Feldman, Emil C. Fischer, Tammy J. Dwyer, Floyd E. Romesberg. Progress Toward a Semi-Synthetic Organism with an Unrestricted Expanded Genetic Alphabet. Journal of the American Chemical Society 2018, 140 (47) , 16115-16123. https://doi.org/10.1021/jacs.8b08416
  9. Tanay Debnath, G. Andrés Cisneros. Investigation of the stability of D5SIC-DNAM-incorporated DNA duplex in Taq polymerase binary system: a systematic classical MD approach. Physical Chemistry Chemical Physics 2024, 26 (9) , 7287-7295. https://doi.org/10.1039/D3CP05571J
  10. Bianbian Huo, Chao Wang, Xiaoqi Hu, Honglei Wang, Gongming Zhu, Anlian Zhu, Lingjun Li. Peripheral substitution effects on unnatural base pairs: A case of brominated TPT3 to enhance replication fidelity. Bioorganic Chemistry 2023, 140 , 106827. https://doi.org/10.1016/j.bioorg.2023.106827
  11. Robert Dörrenhaus, Philip K. Wagner, Stephanie Kath-Schorr. Two are not enough: synthetic strategies and applications of unnatural base pairs. Biological Chemistry 2023, 404 (10) , 883-896. https://doi.org/10.1515/hsz-2023-0169
  12. Honglei Wang, Wuyuan Zhu, Chao Wang, Xiaohuan Li, Luying Wang, Bianbian Huo, Hui Mei, Anlian Zhu, Guisheng Zhang, Lingjun Li. Locating, tracing and sequencing multiple expanded genetic letters in complex DNA context via a bridge-base approach. Nucleic Acids Research 2023, 51 (9) , e52-e52. https://doi.org/10.1093/nar/gkad218
  13. Indu Negi, Bimaldeep Singh, Amanpreet Singh Mahmi, Purshotam Sharma. Structural Properties of Hachimoji Nucleic Acids and Their Building Blocks: Comparison of Genetic Systems with Four, Six and Eight Alphabets. ChemPhysChem 2023, 24 (5) https://doi.org/10.1002/cphc.202200714
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  17. Floyd E. Romesberg. Creation, Optimization, and Use of Semi-Synthetic Organisms that Store and Retrieve Increased Genetic Information. Journal of Molecular Biology 2022, 434 (8) , 167331. https://doi.org/10.1016/j.jmb.2021.167331
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  • Abstract

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    Figure 1

    Figure 1. The dNaM-d5SICS and dNaM-dTPT3 UBPs.

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    Figure 2

    Figure 2. Graphical representation of screen workflow. For sequence contexts 1–4, X denotes NaM or an NaM analog and Y denotes TPT3 or a TPT3 analog. Retention levels of dNaM-dTPT3 are indicated with “+” symbols. See ref 14 for original report of dNaM-dTPT3 retention data.

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    Figure 3

    Figure 3. Structure of analogs used in current study. Shading denotes differences in retention observed in phase 1 of the screen. (A) dNaMTP analogs. Blue shading corresponds to analogs that at 125 μM showed retentions of greater than 90%; green, 50% to 90%; yellow, 20% to 50%; red, <20%. (B) dTPT3TP analogs. Blue shading corresponds to those that at 10 μM showed retentions greater than 90%; red, <10%. Ribose and phosphates omitted for clarity. For original references see Supporting Information.

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    Figure 4

    Figure 4. UBP retention (%). (A) dNaMTP analogs added at either 125 μM or 10 μM and dTPT3TP added at 25 μM. (B) dTPT3TP analogs added at either 125 μM or 10 μM and dNaMTP added at 125 μM. (C) Selected analogs screened against each other with dNaMTP analogs added at 25 μM and dTPT3TP analogs added at 10 μM. Data are an average of three independent trials, with error bars indicating the standard deviation. A single asterisk indicates that no cell growth was observed at the higher concentration, and a double asterisk indicates that no cell growth was observed at either concentration.

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    Figure 5

    Figure 5. UBP retention (%) with dTPT3TP and different dNaMTP analogs added at varying concentrations to the media. Shading indicates the level of UBP retention. Values are the average and standard deviation of three independent determinations.

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    Figure 6

    Figure 6. UBP retention (%) with 25 μM dTPT3TP and varying concentrations of dNaMTP or dCNMOTP added to the media. Shading indicates the level of UBP retention. Values are the average and standard deviation of three independent determinations.

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    Figure 7

    Figure 7. Four optimal UBPs discovered in this work.

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  • References

    This article references 26 other publications.

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      Expansion of the genetic alphabet with an unnatural base pair is a long-standing goal of synthetic biol. The authors have developed a class of unnatural base pairs, formed between d5SICS and analogs of dMMO2 that are efficiently and selectively replicated by the Klenow fragment (Kf) DNA polymerase. In an effort to further characterize and optimize replication, the authors report the synthesis of five new dMMO2 analogs bearing different substituents designed to be oriented into the developing major groove and an anal. of their insertion opposite d5SICS by Kf and Thermus aquaticus DNA polymerase I (Taq). We also expand the anal. of the previously optimized pair, dNaM-d5SICS, to include replication by Taq. Finally, the efficiency and fidelity of PCR amplification of the base pairs by Taq or Deep Vent polymerases was examd. The resulting structure-activity relationship data suggest that the major determinants of efficient replication are the minimization of desolvation effects and the introduction of favorable hydrophobic packing, and that Taq is more sensitive than Kf to structural changes. In addn., the authors identify an analog (dNMO1) that is a better partner for d5SICS than any of the previously identified dMMO2 analogs with the exception of dNaM. They also found that dNaM-d5SICS is replicated by both Kf and Taq with rates approaching those of a natural base pair.
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      A semi-synthetic organism with an expanded genetic alphabet
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