Optimization of an unnatural base pair toward natural-like replication.
Optimization of an unnatural base pair toward natural-like replication.
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DOI：
10.1021/ja807853m
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发表时间：
2009-03-11
影响因子：
15
通讯作者：
Romesberg FE
中科院分区：
文献类型：
--
作者：
Seo YJ;Hwang GT;Ordoukhanian P;Romesberg FE
在双链 DNA 内以及在聚合酶介导的复制过程中选择性配对的主要疏水性非天然核苷酸最近作为扩展遗传字母表的基石而受到广泛关注。我们最近报告了筛选结果和随后的先导命中优化，从而鉴定了核苷酸 dMMO2 和 d5SICS 之间形成的非天然碱基对。这种非天然碱基对由大肠杆菌 DNA 聚合酶 I 的 Klenow 片段复制，比文献中报道的其他候选碱基对具有更好的效率和保真度。然而，它的复制效率仍然明显低于天然碱基对，并且其实际应用需要进一步优化。为了更好地理解和优化非天然碱基对复制的最慢步骤，即在 d5SICS 对面插入 dMMO2，我们合成了两种 dMMO2 衍生物 d5FM 和 dNaM，它们在形状、疏水性和极化性方面与母体核碱基不同。我们发现这两种衍生物比 dMMO2 更有效地插入到 d5SICS 对面，并且总体而言，相应的非自然碱基对通常比 dMMO2 和 d5SICS 之间的碱基对以更高的效率和保真度进行复制。事实上，在 dNaM 和 d5SICS 异质对的情况下，每个单独复制步骤的效率接近自然碱基对的效率，最低总体保真度范围为 103 到 104。此外，这些数据使我们能够提出非自然碱基对复制的通用模型，这将有助于进一步优化非自然碱基对，并可能有助于设计以真正类似自然的效率和保真度复制的其他非自然碱基对。
Predominantly hydrophobic unnatural nucleotides that selectively pair within duplex DNA as well as during polymerase-mediated replication have recently received much attention as the cornerstone of efforts to expand the genetic alphabet. We recently reported the results of a screen and subsequent lead hit optimization that led to the identification of the unnatural base pair formed between the nucleotides dMMO2 and d5SICS. This unnatural base pair is replicated by the Klenow fragment of E. coli DNA polymerase I with better efficiency and fidelity than other candidates reported in the literature. However, its replication remains significantly less efficient than a natural base pair, and further optimization is necessary for its practical use. To better understand and optimize the slowest step of replication of the unnatural base pair, the insertion of dMMO2 opposite d5SICS, we synthesized two dMMO2 derivatives, d5FM and dNaM, which differ from the parent nucleobase in terms of shape, hydrophobicity, and polarizability. We find that both derivatives are inserted opposite d5SICS more efficiently than dMMO2 and that overall the corresponding unnatural base pairs are generally replicated with higher efficiency and fidelity than the pair between dMMO2 and d5SICS. In fact, in the case of the dNaM and d5SICS heteropair, the efficiency of each individual step of replication approaches that of a natural base pair, and the minimum overall fidelity ranges from 103 to 104. In addition, the data allow us to propose a generalized model of unnatural base pair replication, which should aid in the further optimization of the unnatural base pair, and possibly in the design of additional unnatural base pairs that are replicated with truly natural-like efficiency and fidelity.
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影响因子：
64.8
作者：
Matray, TJ;Kool, ET
通讯作者：
Kool, ET
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通讯作者：
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