Division of Biological Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui 230027, PR China.
MOE International Joint Research Laboratory on Synthetic Biology and Medicines, School of Biology and Biological Engineering, South China University of Technology, Guangzhou 510006, PR China.
J Mol Biol. 2024 Oct 15;436(20):168744. doi: 10.1016/j.jmb.2024.168744. Epub 2024 Aug 13.
DNA modified with C2'-methoxy (C2'-OMe) greatly enhances its resistance to nucleases, which is beneficial for the half-life of aptamers and DNA nanomaterials. Although the unnatural DNA polymerases capable of incorporating C2'-OMe modified nucleoside monophosphates (C2'-OMe-NMPs) were engineered via directed evolution, the detailed molecular mechanism by which an evolved DNA polymerase recognizes C2'-OMe-NTPs remains poorly understood. Here, we present the crystal structures of the evolved Stoffel fragment of Taq DNA polymerase SFM4-3 processing the C2'-OMe-GTP in different states. Our results reveal the structural basis for recognition of C2'-methoxy by SFM4-3. Based on the analysis of other mutated residues in SFM4-3, a new Stoffel fragment variant with faster catalytic rate and stronger inhibitor-resistance was obtained. In addition, the capture of a novel pre-insertion co-existing with template 5'-overhang stacking conformation provides insight into the catalytic mechanism of Taq DNA polymerase.
用 C2'-甲氧基（C2'-OMe）修饰的 DNA 极大地提高了其对核酸酶的抗性，这有利于适体和 DNA 纳米材料的半衰期。尽管通过定向进化工程设计了能够掺入 C2'-OMe 修饰的核苷单磷酸（C2'-OMe-NMP）的非天然 DNA 聚合酶，但进化的 DNA 聚合酶识别 C2'-OMe-NTP 的详细分子机制仍知之甚少。在这里，我们展示了 Stoffel 片段的 Taq DNA 聚合酶 SFM4-3 在不同状态下处理 C2'-OMe-GTP 的晶体结构。我们的结果揭示了 SFM4-3 识别 C2'-甲氧基的结构基础。基于对 SFM4-3 中其他突变残基的分析，获得了一种具有更快催化速率和更强抑制剂抗性的新型 Stoffel 片段变体。此外，新型预插入物与模板 5'-突出碱基堆积构象的共存的捕获提供了对 Taq DNA 聚合酶催化机制的深入了解。
