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Chemical Stabilization of Unnatural Nucleotide Triphosphates for the in Vivo Expansion of the Genetic Alphabet
非天然核苷酸三磷酸盐的化学稳定化，用于遗传字母表的体内扩增
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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, 6, 2464–2467
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https://doi.org/10.1021/jacs.6b12731
Published February 7, 2017
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Abstract 抽象

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We have developed an unnatural base pair (UBP) and a semisynthetic organism (SSO) that imports the constituent unnatural nucleoside triphosphates and uses them to replicate DNA containing the UBP. However, propagation of the UBP is at least in part limited by the stability of the unnatural triphosphates, which are degraded by cellular and secreted phosphatases. To circumvent this problem, we now report the synthesis and evaluation of unnatural triphosphates with their β,γ-bridging oxygen replaced with a difluoromethylene moiety, yielding dNaMTPCF2 and dTPT3TPCF2. We find that although dNaMTPCF2 cannot support in vivo replication, likely due to poor polymerase recognition, dTPT3TPCF2 can, and moreover, its increased stability can contribute to increased UBP retention. The data demonstrate the promise of this chemical approach to SSO optimization, and suggest that other modifications should be sought that confer phosphatase resistance without interfering with polymerase recognition.
我们开发了一种非天然碱基对 （UBP） 和一种半合成生物体 （SSO），它们进口组成非天然核苷三磷酸盐，并使用它们来复制含有 UBP 的 DNA。然而，UBP 的传播至少在一定程度上受到非天然三磷酸盐稳定性的限制，这些三磷酸盐被细胞和分泌的磷酸酶降解。为了规避这个问题，我们现在报道了非天然三磷酸盐的合成和评估，其 β，γ 桥氧被二氟亚甲基部分取代，产生 dNaMTPCF2 和 dTPT3TPCF2。我们发现，虽然 dNaMTPCF2 不能支持体内复制，可能是由于聚合酶识别差，但 dTPT3TPCF2 可以，而且，其增加的稳定性有助于提高 UBP 保留。数据证明了这种化学方法对 SSO 优化的前景，并建议应寻求其他修饰，以在不干扰聚合酶识别的情况下赋予磷酸酶抗性。

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Introduction 介绍

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The natural genetic alphabet, conserved throughout nature, is composed of four nucleotide “letters” that pair by complementary hydrogen bonding to provide two base pairs, and these two pairs alone underlie the storage and retrieval of biological information. Significant effort has been directed toward the development of synthetic nucleotides that selectively pair and thus constitute an unnatural base pair (UBP), and three distinct design strategies have yielded UBPs that are well retained in DNA during PCR amplification. (1-3) Hirao and Benner have elegantly used these in vitro expanded genetic alphabets to select for unnatural aptamers with improved properties relative to their fully natural counterparts. (3-5) Although these UBPs were developed by modification of the shape or hydrogen-bonding patterns of natural nucleobases, our approach instead relied on synthetic nucleotides with nucleobases that have little similarity to their natural counterparts and that interact via hydrophobic and packing forces; (6) an approach that eventually culminated in the identification of a family of UBPs represented by dNaM-d5SICS (7) and dNaM-dTPT3 (8, 9) (Figure 1A). Despite having little to no nucleobase homology with their natural counterparts, these UBPs are efficiently replicated via a unique, mutually induced-fit mechanism. (6, 10, 11)
自然遗传字母表在整个自然界中都是保守的，由四个核苷酸“字母”组成，它们通过互补的氢键配对以提供两个碱基对，而这两对字母是生物信息存储和检索的基础。人们已经投入了大量精力来开发选择性配对的合成核苷酸，从而构成非天然碱基对 （UBP），并且三种不同的设计策略已经产生了在 PCR 扩增过程中很好地保留在 DNA 中的 UBP。（1-3） Hirao 和 Benner 优雅地使用这些体外扩展的遗传字母来选择相对于完全天然的适配子具有改进特性的非天然适配子。（3-5） 尽管这些 UBP 是通过修改天然核碱基的形状或氢键模式开发的，但我们的方法反而依赖于合成核苷酸，这些核苷酸的核碱基与其天然对应物几乎没有相似性，并且通过疏水和堆积力相互作用;（6） 一种方法最终确定了以 dNaM-d 5SICS （7） 和 dNaM-d TPT3 （8， 9） 为代表的 UBP 家族（图 1A）。尽管与天然对应物几乎没有核碱基同源性，但这些 UBP 通过独特的互诱导拟合机制进行有效复制。（6、10、11）

Figure 1 图 1

Figure 1. Unnatural nucleotides. (A) dNaM-d5SICS and dNaM-dTPT3 UBPs. (B) dTPT3TPCF2 and dNaMTPCF2, with α, β, and γ phosphates labeled.
图 1.非天然核苷酸。（A） dNaM-d 5SICS 和 dNaM-d TPT3 UBP。（B） dTPT3TPCF2 和 dNaMTPCF2，标记了 α、β 和 γ 磷酸盐。

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As opposed to in vitro applications, our efforts have focused on the use of the UBPs as the foundation of a semisynthetic organism (SSO) that stably stores and retrieves increased information. We recently demonstrated that Escherichia coli grown in media supplemented with the unnatural triphosphates dNaMTP and d5SICSTP, and provided with the nucleoside triphosphate transporter PtNTT2 to import them, is able to replicate a plasmid containing the dNaM-d5SICS UBP. (12) However, a significant limitation of the nascent SSO is UBP loss with extended or high density growth, which appears to result, at least in part, from the dephosphorylation of the unnatural triphosphates, reducing their concentration below that required for efficient replication. (12) Although recent efforts have demonstrated that the dNaM-dTPT3 UBP is retained in the SSO better than the originally employed dNaM-d5SICS UBP, (13) dTPT3TP is likely also susceptible to the same phospholytic degradation. Efforts to inhibit degradation or identify and delete the genes that encode the phosphatases responsible have so far been unsuccessful (our unpublished results and ref 12), but suggest that multiple phosphatases may be involved.
与体外应用相反，我们的工作重点是使用 UBP 作为半合成生物体 （SSO） 的基础，该生物体可以稳定地存储和检索增加的信息。我们最近证明，在补充有非天然三磷酸盐 dNaMTP 和 d5SICSTP 的培养基中生长的大肠杆菌，并提供核苷三磷酸转运蛋白 PtNTT2 以输入它们，能够复制含有 dNaM-d 5SICS UBP 的质粒。（12） 然而，新生 SSO 的一个显着限制是 UBP 在延长或高密度生长时丢失，这似乎至少部分是由于非天然三磷酸盐的去磷酸化，将其浓度降低到有效复制所需的水平以下。（12） 尽管最近的工作表明 dNaM-d TPT3 UBP 比最初使用的 dNaM-d 5SICS UBP 更好地保留在 SSO 中，但 （13） dTPT3TP 也可能容易受到相同的磷酸降解。到目前为止，抑制降解或识别和删除编码负责磷酸酶的基因的努力尚未成功（我们未发表的结果和参考文献 12），但表明可能涉及多种磷酸酶。
Although a potential solution to the problem of unnatural triphosphate degradation would be to add them continuously during growth, this would be wasteful and would complicate the use of the SSO. In contrast, chemical stabilization of the triphosphates would be more practical. To explore this chemical approach, we synthesized analogs of the unnatural triphosphates with their β,γ-bridging oxygen atom replaced with a difluoromethylene moiety, yielding dNaMTPCF2 and dTPT3TPCF2 (Figure 1B). The data reveal that although the β,γ-CF2 modification imparts stability to both triphosphates, it also impacts transporter and polymerase recognition in a nucleobase-dependent manner. The effects on polymerase recognition appear larger and preclude the use of dNaMTPCF2, but not dTPT3TPCF2, suggesting that it or other similarly stabilized unnatural triphosphates should be useful in the effort to expand the genetic alphabet of a living organism.
尽管非自然三磷酸盐降解问题的潜在解决方案是在生长过程中不断添加它们，但这将是浪费，并且会使 SSO 的使用复杂化。相比之下，三磷酸盐的化学稳定化会更实用。为了探索这种化学方法，我们合成了非天然三磷酸盐的类似物，其 β，γ 桥氧原子被二氟亚甲基部分取代，产生 dNaMTPCF2 和 dTPT3TPCF2 （图 1B）。数据显示，尽管 β，γ-CF2 修饰赋予两种三磷酸盐稳定性，但它也以核碱基依赖性方式影响转运蛋白和聚合酶的识别。对聚合酶识别的影响似乎更大，并且排除了 dNaMTPCF2 的使用，但排除了 dTPT3TPCF2 的使用，这表明它或其他类似稳定的非天然三磷酸盐应该有助于扩展生物体的遗传字母表。

Experimental Section 实验部分

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General 常规

For synthetic procedures, all reactions were carried out in oven-dried glassware under inert atmosphere, and all solvents were distilled and/or dried over 4 Å molecular sieves. All other chemical reagents were purchased from Aldrich, unless otherwise noted. 1H and 13C spectra were recorded on a Bruker DPX 400 mHz NMR instrument equipped with a QNP probe. 31P and 19F NMR spectra were recorded on a Bruker AVIII HD 600 mHz NMR instrument equipped with a CPDCH cryoprobe. Mass spectroscopic data were obtained from the core facilities at The Scripps Research Institute.
对于合成程序，所有反应均在惰性气氛下在烘箱干燥的玻璃器皿中进行，所有溶剂均在 4 Å 分子筛上蒸馏和/或干燥。除非另有说明，否则所有其他化学试剂均购自 Aldrich。1H 和 13C 波谱在配备 QNP 探头的布鲁克 DPX 400 mHz NMR 仪器上记录。31P 和 19F NMR 波谱在配备 CPDCH 低温探头的布鲁克 AVIII HD 600 mHz NMR 仪器上记录。质谱数据来自斯克里普斯研究所的核心设施。
All bacterial cultures were grown in 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). All experiments involving bacterial growth were conducted in microwell plates (96-well or 48-well, as needed). When noted, antibiotics were used at the following concentrations: chloramphenicol, 5 μg/mL; ampicillin, 50 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 or dTPT3 were synthesized by Biosearch Technologies (Petaluma, CA) with purification by reverse phase cartridge and kindly provided by Synthorx (La Jolla, CA).
所有细菌培养物均在补充有磷酸钾 （50 mM，pH 7） 的液体 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）中生长。所有涉及细菌生长的实验均在微孔板（根据需要为 96 孔或 48 孔）中进行。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，50 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 或 dTPT3 的寡核苷酸由 Biosearch Technologies（加利福尼亚州佩塔卢马）合成，通过反相小柱纯化，由 Synthorx（加利福尼亚州拉霍亚）友情提供。

Degradation of Nucleotide Triphosphates
核苷酸三磷酸盐的降解

An overnight culture (∼3 mL) of E. coli BL21(DE3) lacZYA::CmR (13) was diluted 100-fold into fresh media and incubated at 37 °C for 2 h. At this time, cells were diluted back to an OD600 of 0.10 in 500 μL media containing 200 mM nucleotide triphosphate. Cultures were incubated with the triphosphates at 37 °C with shaking, and at designated times samples were collected by removing an aliquot (50 μL) and immediately pelleting the cell fraction (9,000 r.c.f. for 5 min, 4 °C). An aliquot (40 μL) of the supernatant (i.e., media fraction) was removed and mixed with acetonitrile (80 μL) and then incubated at room temperature (∼23 °C) for 30 min before pelleting (12,000 r.c.f. for 10 min, RT) to remove precipitate. The supernatant was collected, dried (SpeedVac), resuspended in 20 μL of 0.1 M triethylammonium bicarbonate (TEAB) pH 7.5, and then analyzed by HPLC (Agilent 1100 series) using a Phenomenex Jupiter LC column (3 mm C18 300 Å, 250 × 4.6 mm) as described previously. (12) Percent remaining triphosphate was determined by integrating the area under the curve (AUC) for each species present (i.e., triphosphate, diphosphate, monophosphate), and dividing the AUC of the triphosphate peak by the sum of the three peaks.
将大肠杆菌 BL21（DE3） lacZYA：：CmR （13） 的过夜培养物 （∼3 mL） 稀释到新鲜培养基中 100 倍，并在 37 °C 下孵育 2 小时。此时，在含有 200 mM 核苷酸三磷酸的 500 μL 培养基中将细胞稀释回 OD600 0.10。将培养物与三磷酸盐在 37 °C 下振荡孵育，并在指定时间通过取出等分试样 （50 μL） 并立即沉淀细胞组分（9,000 r.c.f.，5 分钟，4 °C）来收集样品。除去等分试样 （40 μL） 上清液（即培养基组分）并与乙腈 （80 μL） 混合，然后在室温 （∼23 °C） 下孵育 30 分钟，然后沉淀（12,000 r.c.f.，10 分钟，RT）以去除沉淀物。收集上清液，干燥 （SpeedVac），重悬于 20 μL 0.1 M 三乙基碳酸氢铵 （TEAB） （pH 7.5） 中，然后如前所述使用 Phenomenex Jupiter 液相色谱柱（3 mm C18 300 Å，250 × 4.6 mm）通过 HPLC（Agilent 1100 系列）进行分析。（12） 通过将存在的每种物质（即三磷酸盐、二磷酸盐、单磷酸盐）的曲线下面积 （AUC） 积分，并将三磷酸盐峰的 AUC 除以三个峰的总和来确定剩余三磷酸盐的百分比。

Kinetic Assay 动力学测定

Steady-state kinetic assays were performed as described previously (14) with oligonucleotide primers and templates described in Table S1. The reaction products were resolved by denaturing PAGE, and the resulting gel was imaged on a Typhoon 9210 flatbed laser scanner (GE Healthcare). Image Studio Lite (LI-COR Biosciences) was used to quantify band density of imaged gels. Kinetic parameters (KM and Vmax) were determined by plotting the observed rate of incorporation against triphosphate concentration and fitting the data to the Michaelis–Menten equation. The reported values are the average and standard deviation of at least three independent determinations.
如前所述 （14） 使用表 S1 中描述的寡核苷酸引物和模板进行稳态动力学测定。通过变性 PAGE 分离反应产物，并在 Typhoon 9210 平板激光扫描仪 （GE Healthcare） 上对所得凝胶进行成像。Image Studio Lite （LI-COR Biosciences） 用于定量成像凝胶的条带密度。动力学参数 （K、M 和 Vmax） 是通过绘制观察到的掺入率与三磷酸盐浓度的关系并将数据拟合到 Michaelis-Menten 方程来确定的。报告的值是至少三个独立测定的平均值和标准差。

Inhibition-Based Uptake Assay
基于抑制的摄取测定

An overnight culture (∼3 mL) of the E. coli SSO YZ3 (13) was diluted 100-fold in media supplemented with chloramphenicol (∼8 mL) and incubated at 37 °C to an OD600 ∼ 0.4–0.6 (∼1.5 h). To an aliquot of cells (92.5 μL) was added a solution of an unnatural triphosphate (500 μM), ATP (50 μM), and [α-32P]-ATP (4 μCi/mL, PerkinElmer), to a final volume of 100 μL. Each sample was run in parallel with an aliquot of cells receiving no unnatural triphosphate as an ATP-only control. Treated cells were incubated at 37 °C for 10 min, collected through a 96-well 0.65 mm glass fiber filter plate (MultiScreen, EMD Millipore) under vacuum, and then washed with ddH2O (2 × 100 μL). Filters were then exposed overnight to a storage phosphor screen, which was then imaged as described above. Inhibition of ATP uptake was determined by densitometric analysis of the resulting image, normalizing radioactive density to the ATP-only control and expressing the value as percent inhibition of ATP uptake. Reported values are the average and standard deviation of at least three independent determinations.
将大肠杆菌SSO YZ3 （13）的过夜培养物（∼3 mL）在补充有氯霉素（∼8 mL）的培养基中稀释100倍，并在37 °C下孵育至OD600 ∼ 0.4–0.6 （∼1.5 h）。向等分的细胞 （92.5 μL） 中加入非天然三磷酸盐 （500 μM）、ATP （50 μM） 和 [α-32P]-ATP（4 μCi/mL，珀金埃尔默）的溶液，最终体积为 100 μL。每个样品与等分试样平行运行，这些细胞接受无非天然三磷酸盐作为仅 ATP 对照。将处理过的细胞在 37 °C 下孵育 10 分钟，在 96 孔 0.65 mm 玻璃纤维过滤板（MultiScreen，EMD Millipore）真空下收集，然后用 ddH2O （2 × 100 μL） 洗涤。然后将滤光片暴露在存储荧光屏上过夜，然后按上述方式成像。通过对所得图像进行密度分析来确定 ATP 摄取的抑制，将放射性密度标准化为仅 ATP 对照，并将该值表示为 ATP 摄取的抑制百分比。报告的值是至少三个独立测定的平均值和标准差。

Analysis of in Vivo Replication
体内复制分析

Plasmids containing the UBP were prepared and used to transform the SSO as described previously. (12, 13) After recovery, cells were diluted into media containing different combination and concentrations of dNaMTP and dTPT3 or their β,γ-CF2 modified counterparts. Plasmids were recovered at defined times and analyzed for retention of the UBP as described previously (12, 13) and in the Supporting Information.
如前所述制备含有 UBP 的质粒并用于转化 SSO。（12， 13）回收后，将细胞稀释到含有不同组合和浓度的 dNaMTP 和 dTPT3 或其 β，γ-CF2 修饰对应物的培养基中。在规定的时间回收质粒，并如前所述 （12， 13） 和支持信息中分析 UBP 的保留。

Results and Discussion 结果与讨论

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To explore the chemical stabilization of the unnatural triphosphates, we synthesized dNaMTPCF2 and dTPT3TPCF2. Although the β,γ-CF2 modification may impact polymerase binding, its electronegativity should favor nucleophilic attack at the a phosphate, (15-18) while eliminating phospholytic cleavage between the β and γ phosphates, likely the major route of degradation. Briefly, the free nucleosides dNaM and dTPT3 were synthesized as reported previously, (14, 19) and then, using the method reported by McKenna et al., (20) they were converted to dNaMTPCF2 and dTPT3TPCF2.
为了探索非天然三磷酸盐的化学稳定性，我们合成了 dNaMTPCF2 和 dTPT3TPCF2。尽管 β，γ-CF2 修饰可能会影响聚合酶结合，但其电负性应有利于磷酸盐 a 的亲核攻击 （15-18），同时消除 β 磷酸盐和 γ 磷酸盐之间的磷酸裂解，这可能是降解的主要途径。简而言之，游离核苷 dNaM 和 dTPT3 按照先前报道的 （14， 19） 合成，然后，使用 McKenna 等人报道的方法 （20） 将它们转化为 dNaMTPCF2 和 dTPT3TPCF2。
With these analogs in hand, we first tested their extracellular stability in cultures of actively growing bacteria. E. coli BL21(DE3) was grown to early log phase (OD600 ∼ 0.1), at which time 200 μM of a modified or unmodified unnatural triphosphate was added. Following incubation at 37 °C, cells were removed by centrifugation, soluble proteins in the recovered growth medium were precipitated, and the amount of intact triphosphate was determined by HPLC (Figure 2A). Without modification, no triphosphate was detected after 24 h, consistent with rapid degradation, presumably by secreted phosphatases. However, no degradation of either dNaMTPCF2 or dTPT3TPCF2 was apparent, confirming that the β,γ-CF2 moiety significantly stabilizes the triphosphates under conditions employed to grow the SSO.
有了这些类似物，我们首先在活跃生长的细菌培养物中测试了它们的细胞外稳定性。大肠杆菌BL21（DE3） 生长到早期对数期 （OD600 ∼ 0.1），此时加入 200 μM 修饰或未修饰的非天然三磷酸盐。在 37 °C 下孵育后，通过离心除去细胞，沉淀回收的生长培养基中的可溶性蛋白质，并通过 HPLC 测定完整三磷酸盐的量（图 2A）。在没有修饰的情况下，24 小时后未检测到三磷酸盐，这与快速降解一致，可能是由分泌的磷酸酶引起的。然而，dNaMTPCF2 或 dTPT3TPCF2 的降解均不明显，证实了 β，γ-CF2 部分在用于生长 SSO 的条件下显着稳定了三磷酸盐。

Figure 2 图 2

Figure 2. Extracellular stability and transporter recognition of modified and unmodified unnatural triphosphates. (A) β,γ-CF2 modification stabilizes the unnatural triphosphates to degradation by secreted phosphatases. (B) β,γ-CF2 modified unnatural triphosphates are still recognized by the SSO’s PtNTT2 transporter as detected by the inhibition of [α-32P]ATP uptake (see text and Supporting Information for details).
图 2.修饰和未修饰的非天然三磷酸盐的细胞外稳定性和转运蛋白识别。（A） β，γ-CF2 修饰使非天然三磷酸盐稳定，使其被分泌的磷酸酶降解。（B） β，γ-CF2 修饰的非天然三磷酸盐仍被 SSO 的 PtNTT2 转运蛋白识别，通过抑制 [α-32P] ATP 摄取来检测（有关详细信息，请参见文本和支持信息）。

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To determine whether β,γ-CF2 modification is compatible with polymerase-mediated replication, we next performed steady-state kinetic assays with the Klenow fragment of E. coli DNA polymerase I (Supporting Information), which served as a model polymerase during UBP development. Even under forcing conditions, we were unable to detect any insertion of dNaMTPCF2 opposite dTPT3. In contrast, we found that the polymerase does insert dTPT3TPCF2 opposite dNaM; however, although it does so with a reasonable KM of 15.7 ± 7.5 nM, the kcat of 1.4 ± 0.5 min–1 is somewhat less than that observed for dTPT3TP (our unpublished results). This data suggests that dNaMTPCF2 might not be compatible with replication within the SSO, and that while replication may occur with dTPT3TPCF2, the β,γ-CF2 moiety may lower its efficiency.
为了确定 β，γ-CF2 修饰是否与聚合酶介导的复制兼容，我们接下来使用大肠杆菌 DNA 聚合酶 I 的 Klenow 片段（支持信息）进行了稳态动力学测定，该片段在 UBP 开发过程中用作模型聚合酶。即使在强迫条件下，我们也无法检测到 dNaMTPCF2 与 dTPT3 相对的任何插入。相比之下，我们发现聚合酶确实在 dNaM 的对面插入 dTPT3TPCF2;然而，尽管它的合理 KM 为 15.7 ± 7.5 nM，但 1.4 ± 0.5 min–1 的 kcat 略低于观察到的 dTPT3TP（我们未发表的结果）。该数据表明 dNaMTPCF2 可能与 SSO 内的复制不兼容，虽然 dTPT3TPCF2 可能会发生复制，但 β，γ-CF2 部分可能会降低其效率。
For use with the SSO, the stabilized unnatural triphosphates must first be recognized as a substrate by PtNTT2 and imported into the cell. To begin to examine recognition by PtNTT2, we characterized the ability of each modified and unmodified analog to decrease the import of [α-32P]ATP due to competitive inhibition, as previously described. (21) The addition of 500 mM of either dNaMTPCF2 or dTPT3TPCF2 inhibited the uptake of [α-32P]ATP (Figure 2B and Supporting Information). Although the level of inhibition was somewhat less than with dNaMTP and dTPT3TP, the data suggests that CF2 modification does not fully ablate recognition by the SSO’s transporter.
为了与 SSO 一起使用，稳定的非天然三磷酸盐必须首先被 PtNTT2 识别为底物并导入细胞。为了开始检查 PtNTT2 的识别，我们表征了每种修饰和未修饰的类似物由于竞争性抑制而减少 [α-32P] ATP 输入的能力，如前所述。（21） 添加 500 mM 的 dNaMTPCF2 或 dTPT3TPCF2 抑制了 [α-32 P] ATP 的摄取（图 2B 和支持信息）。尽管抑制水平略低于 dNaMTP 和 dTPT3TP，但数据表明 CF2 修饰并不能完全消融 SSO 转运蛋白的识别。
We next proceeded to examine whether dNaMTPCF2 and/or dTPT3TPCF2 could support replication of the dNaM-dTPT3 UBP within the SSO. A pUC19 plasmid containing a single dNaM-dTPT3 UBP embedded within the TK1 sequence, (12) was constructed via Golden Gate assembly, and used to transform the SSO. Cells were grown to an OD600 of ∼0.7 in media supplemented with 3.0 μM dTPT3TP and 250 μM dNaMTP, or with one of the unnatural triphosphates replaced with its CF2-modified analog at the same concentration, and plasmids were recovered and analyzed for UBP retention (Figure 3 and Supporting Information). Under these relatively permissive conditions, as expected, providing dNaMTP and dTPT3TP in the media resulted in high UBP retention (99.1 ± 2.1%). Although retention was undetectable when dNaMTPCF2 and dTPT3TP were provided, it was significant when dNaMTP and dTPT3TPCF2 were provided (86.0 ± 11.4%). When combined with the data described above, this suggests that both of the modified unnatural triphosphates are imported, but dNaMTPCF2 is not recognized during replication, and while dTPT3TPCF2 is recognized, it is recognized with somewhat reduced fidelity than its unmodified counterpart.
接下来，我们继续检查 dNaMTPCF2 和/或 dTPT3TPCF2 是否可以支持 SSO 中 dNaM-d TPT3 UBP 的复制。通过 Golden Gate 组装构建含有嵌入 TK1 序列中的单个 dNaM-d TPT3 UBP 的 pUC19 质粒 （12），并用于转化 SSO。在补充有 3.0 μMd TPT3TP 和 250 μMd NaMTP 的培养基中，或用相同浓度的 CF2 修饰类似物替换其中一种非天然三磷酸盐，将细胞生长至 OD600 为 ∼0.7，并回收质粒并分析 UBP 保留（图 3 和支持信息）。在这些相对宽松的条件下，正如预期的那样，在培养基中提供 dNaMTP 和 dTPT3TP 会导致高 UBP 保留率 （99.1 ± 2.1%）。虽然当提供 dNaMTPCF2 和 dTPT3TP 时检测不到保留，但当提供 dNaMTP 和 dTPT3TPCF2 时，保留率很高 （86.0 ± 11.4%）。当与上述数据结合时，这表明两种修饰的非天然三磷酸盐都是输入的，但在复制过程中无法识别 dNaMTPCF2，而虽然识别了 dTPT3TPCF2，但它的识别保真度比未修饰的对应物略低。

Figure 3 图 3

Figure 3. Streptavidin gel shifts characterizing the extent of UBP retention in vivo. Recovered plasmids were PCR amplified with a dNaMTP analog that is biotin tagged and thus the percentage of the slower migrating (upper) band, relative to the sum of the slower and faster migrating bands represents the percent UBP retention. Note that the faster migrating (unshifted) band sometimes appears as a doublet, but this is observed with the input plasmid, as well, and thus is not related to in vivo replication. The percentage shifted is normalized to that measured for the input plasmid (shown on left). A molecular weight marker is included in the lane labeled “M”. See Supporting Information for details.
图 3.链霉亲和素凝胶变化表征了 UBP 在体内保留的程度。回收的质粒用生物素标记的 dNaMTP 类似物进行 PCR 扩增，因此较慢迁移（上）条带相对于较慢和较快迁移条带之和的百分比表示 UBP 保留百分比。请注意，快速迁移（未移位）条带有时表现为双峰，但这也可以在输入质粒中观察到，因此与体内复制无关。将偏移的百分比标准化为输入质粒测得的百分比（如左图所示）。分子量标记物包含在标记为“M”的泳道中。有关详细信息，请参阅支持信息。

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To explore whether the increased stability of dTPT3TPCF2 can result in increased intracellular concentrations that result in more efficient UBP replication, despite its inherently reduced fidelity, we examined replication of the dNaM-dTPT3 UBP within the SSO under less permissive conditions, growing cells to deep stationary phase (OD600 ∼ 9) in the presence of 0.75 μM dTPT3TP or dTPT3TPCF2, and 250, 1000, or 2000 μM dNaMTP (Figure 3). For dTPT3TP, the level of UBP retention remained relatively constant across the different dNaMTP concentrations, at 72 ± 9%, 83 ± 4%, and 81 ± 9%, respectively. However, with dTPT3TPCF2, UBP retentions were 51 ± 11%, 80 ± 9%, and 85 ± 14%, respectively, with increasing dNaMTP concentrations. It is unclear if the ∼85% retention, which was also observed under the permissive conditions, is significant, perhaps representing the maximal fidelity possible with polymerase saturation. Regardless, it is clear that under these more challenging growth conditions, compensating for dNaMTP degradation by increasing its concentration can increase UBP retention, but this obviously requires that sufficient levels of its cognate triphosphate also be maintained, which appears to be accomplished by CF2-modification.
为了探索 dTPT3TPCF2 稳定性的增加是否会导致细胞内浓度增加，从而导致更有效的 UBP 复制，尽管其固有的保真度降低，我们检查了 dNaM-d TPT3 UBP 在 SSO 内的复制在不太宽松的条件下，细胞生长至深固定相 （OD600 ∼ 9） 在 0.75 μM dTPT3TP 或 dTPT3TP CF2 存在下和 250、1000 或 2000 μM dNaMTP（图 3）。对于 dTPT3TP，不同 dNaMTP 浓度的 UBP 保留水平保持相对恒定，分别为 72 ± 9%、83 ± 4% 和 81 ± 9%。然而，随着 dNaMTP 浓度的增加，dTPT3TPCF2 的 UBP 保留率分别为 51 ± 11%、80 ± 9% 和 85 ± 14%。目前尚不清楚在允许条件下也观察到的 ∼85% 保留率是否显着，可能代表了聚合酶饱和度可能的最大保真度。无论如何，很明显，在这些更具挑战性的生长条件下，通过增加其浓度来补偿 dNaMTP 降解可以增加 UBP 保留，但这显然需要维持其同源三磷酸盐的足够水平，这似乎是通过 CF2 修饰实现的。
The goal of synthetic biology is to impart living organisms with new and unnatural traits and even to create artificial life, (22) and at its heart is the power to draw upon both synthetic chemistry and more biology-focused approaches. Although early SSO development in our laboratory focused on the chemical optimization of the unnatural nucleotides, more recent efforts have focused on the optimization of the cell itself for import and retention of the UBP. The data presented herein reveal that, as expected, the β,γ-CF2 modification of either unnatural triphosphate imparts resistance to degradation, but that it also impacts transporter and polymerase recognition in a manner that depends on the nucleobase. The reduced polymerase recognition of dNaMTPCF2 precludes its use in the SSO, but the stability and at least reasonable uptake and polymerase recognition of dTPT3TPCF2 demonstrate the promise of this chemical approach to SSO optimization. Further modifications should be sought with the goal of stabilizing both unnatural triphosphates and in a manner that does not interfere with uptake or replication.
合成生物学的目标是赋予生物体新的和非自然的特征，甚至创造人工生命，（22），其核心是利用合成化学和更注重生物学的方法的能力。尽管我们实验室的早期 SSO 开发侧重于非天然核苷酸的化学优化，但最近的工作集中在优化细胞本身以导入和保留 UBP。本文提供的数据表明，正如预期的那样，非天然三磷酸盐的 β，γ-CF2 修饰赋予了抗降解性，但它也以依赖于核碱基的方式影响转运蛋白和聚合酶的识别。dNaMTPCF2 的聚合酶识别降低，使其无法在 SSO 中使用，但 dTPT3TPCF2 的稳定性以及至少合理的摄取和聚合酶识别证明了这种化学方法对 SSO 优化的前景。应寻求进一步的修饰，以稳定非天然三磷酸盐，并且以不干扰摄取或复制的方式。

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.6b12731.
支持信息可在 ACS Publications 网站免费获取，网址为 DOI：10.1021/jacs.6b12731。

  • Supporting methods, synthesis, and characterization of dTPT3TPCF2 and dNaMTPCF2 (PDF)
    dTPT3TPCF2 和 dNaMTPCF2 的支持方法、合成和表征 （PDF）

Terms & Conditions  条款和条件

Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.
大多数电子支持信息文件无需订阅 ACS Web 版本即可获得。此类文件可以按文章下载用于研究用途（如果有链接到相关文章的公共使用许可证，则该许可证可能允许其他用途）。可以通过 RightsLink 权限系统（http://pubs.acs.org/page/copyright/permissions.html）请求从 ACS 获得用于其他用途的许可。

Author Information 作者信息

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  • Corresponding Author 通讯作者
  • Authors 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
    • Vivian T. Dien - Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, United States
      Vivian T. Dien - 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.) and National Science Foundation Graduate Research Fellowships (Grant No. NSF/DGE-1346837 to A.W.F.)
这项工作得到了美国国立卫生研究院（F.E.R. 的第 GM060005 号和GM118178号）和美国国家科学基金会研究生研究奖学金（第 1 号）的支持。NSF/DGE-1346837 至 A.W.F.）

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本文引用了其他 22 种出版物。

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  • Abstract

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    Figure 1

    Figure 1. Unnatural nucleotides. (A) dNaM-d5SICS and dNaM-dTPT3 UBPs. (B) dTPT3TPCF2 and dNaMTPCF2, with α, β, and γ phosphates labeled.

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    Figure 2

    Figure 2. Extracellular stability and transporter recognition of modified and unmodified unnatural triphosphates. (A) β,γ-CF2 modification stabilizes the unnatural triphosphates to degradation by secreted phosphatases. (B) β,γ-CF2 modified unnatural triphosphates are still recognized by the SSO’s PtNTT2 transporter as detected by the inhibition of [α-32P]ATP uptake (see text and Supporting Information for details).

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    Figure 3

    Figure 3. Streptavidin gel shifts characterizing the extent of UBP retention in vivo. Recovered plasmids were PCR amplified with a dNaMTP analog that is biotin tagged and thus the percentage of the slower migrating (upper) band, relative to the sum of the slower and faster migrating bands represents the percent UBP retention. Note that the faster migrating (unshifted) band sometimes appears as a doublet, but this is observed with the input plasmid, as well, and thus is not related to in vivo replication. The percentage shifted is normalized to that measured for the input plasmid (shown on left). A molecular weight marker is included in the lane labeled “M”. See Supporting Information for details.

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  • References

    This article references 22 other publications.

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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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      Betz, K.; Malyshev, D.; 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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      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
      11
      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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      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
      12
      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.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXotVyqtb8%253D&md5=97b4b184cda52cc809b1705e5e88ad8e
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      Zhang, Y.; Lamb, B.; Feldman, A. W.; Zhou, A. X.; Lavergne, T.; Li, L.; Romesberg, F. E. Proc. Natl. Acad. Sci. U. S. A. 2017, 201616443 DOI: 10.1073/pnas.1616443114
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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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      Sucato, C. A.; Upton, T. G.; Kashemirov, B. A.; Batra, V. K.; Martinek, V.; Xiang, Y.; Beard, W. A.; Pedersen, L. C.; Wilson, S. H.; McKenna, C. E.; Florian, J.; Warshel, A.; Goodman, M. F. Biochemistry 2007, 46, 461– 471 DOI: 10.1021/bi061517b
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      Sucato, C. A.; Upton, T. G.; Kashemirov, B. A.; Osuna, J.; Oertell, K.; Beard, W. A.; Wilson, S. H.; Florian, J.; Warshel, A.; McKenna, C. E.; Goodman, M. F. Biochemistry 2008, 47, 870– 879 DOI: 10.1021/bi7014162
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      Batra, V. K.; Pedersen, L. C.; Beard, W. A.; Wilson, S. H.; Kashemirov, B. A.; Upton, T. G.; Goodman, M. F.; McKenna, C. E. J. Am. Chem. Soc. 2010, 132, 7617– 7625 DOI: 10.1021/ja909370k
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      Oertell, K.; Chamberlain, B. T.; Wu, Y.; Ferri, E.; Kashemirov, B. A.; Beard, W. A.; Wilson, S. H.; McKenna, C. E.; Goodman, M. F. Biochemistry 2014, 53, 1842– 1848 DOI: 10.1021/bi500101z
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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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      McKenna, C. E.; Kashemirov, B. A.; Upton, T. G.; Batra, V. K.; Goodman, M. F.; Pedersen, L. C.; Beard, W. A.; Wilson, S. H. J. Am. Chem. Soc. 2007, 129, 15412– 15413 DOI: 10.1021/ja072127v
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      Ast, M.; Gruber, A.; Schmitz-Esser, S.; Neuhaus, H. E.; Kroth, P. G.; Horn, M.; Haferkamp, I. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 3621– 3626 DOI: 10.1073/pnas.0808862106
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      Leduc, S. The Mechanisms of Life; Rebman Company: New York, 1911.
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