<br>用 DNA 切割 DNA：铜离子驱动的结构和反应性的协同调节,Advanced Science

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用 DNA 切割 DNA：铜离子驱动的结构和反应性的协同调节
Advanced Science ( IF 14.3 ) Pub Date : 2024-02-28 , DOI: 10.1002/advs.202306710
Sarath Chandra Dantu ₁ , Mahdi Khalil ₂ , Marc Bria ₃ , Christine Saint-Pierre ₄ , Maylis Orio ₅ , Didier Gasparutto ₄ , Giuseppe Sicoli ₂

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通过结合分子动力学 (MD) 模拟和先进的电子顺磁共振（电子自旋共振）EPR/ESR 光谱，对先前通过体外选择分离的铜依赖性自裂解 DNA（DNAzyme 或脱氧核酶）进行了分析，为以下方面提供了见解：裂解反应的结构和机理特征。 MD 模拟中建模的 46 核苷酸脱氧核酶形成双链体和三链体亚结构，侧翼是高度保守的催化核心。 DNA自切割构建体还可以形成具有独特底物和酶结构域的双分子复合物。高度动态的结构与底物的氧化位点特异性裂解相结合是需要阐明的两个关键方面。通过将 EPR/ESR 光谱与选择性同位素标记的核苷酸相结合，可以克服与“金属汤”场景相关的主要缺点，也称为辅助因子与底物的“超化学计量”比率，通常是 DNA 切割所需的这些基于核酸的酶内的反应。这里描述的对内源顺磁中心 (Cu ²⁺ ) 的关注为涉及几种不同辅因子的混合物的分析铺平了道路。此外，在更复杂的结构中插入裂解反应现在是 EPR/ESR 光谱研究适用性的现实视角。

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Cleaving DNA with DNA: Cooperative Tuning of Structure and Reactivity Driven by Copper Ions

A copper-dependent self-cleaving DNA (DNAzyme or deoyxyribozyme) previously isolated by in vitro selection has been analyzed by a combination of Molecular Dynamics (MD) simulations and advanced Electron Paramagnetic Resonance (Electron Spin Resonance) EPR/ESR spectroscopy, providing insights on the structural and mechanistic features of the cleavage reaction. The modeled 46-nucleotide deoxyribozyme in MD simulations forms duplex and triplex sub-structures that flank a highly conserved catalytic core. The DNA self-cleaving construct can also form a bimolecular complex that has a distinct substrate and enzyme domains. The highly dynamic structure combined with an oxidative site-specific cleavage of the substrate are two key-aspects to elucidate. By combining EPR/ESR spectroscopy with selectively isotopically labeled nucleotides it has been possible to overcome the major drawback related to the “metal-soup” scenario, also known as “super-stoichiometric” ratios of cofactors versus substrate, conventionally required for the DNA cleavage reaction within those nucleic acids-based enzymes. The focus on the endogenous paramagnetic center (Cu²⁺) here described paves the way for analysis on mixtures where several different cofactors are involved. Furthermore, the insertion of cleavage reaction within more complex architectures is now a realistic perspective towards the applicability of EPR/ESR spectroscopic studies.

更新日期：2024-02-28

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