PDF(Pubmed)
Abstract：
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.
摘要：
通过分子动力学（MD）模拟和先进的电子顺磁共振（电子自旋共振）EPR/ESR光谱相结合，分析了先前通过体外选择分离的铜依赖性自切割DNA（DNAzyme或脱氧核酶），提供有关裂解反应的结构和机理特征的见解。在MD模拟中建模的46个核苷酸的脱氧核酶形成了双链体和三链体亚结构，它们位于高度保守的催化核心的侧面。DNA自切割构建体还可以形成具有不同底物和酶结构域的双分子复合物。高度动态结构与底物的氧化位点特异性裂解相结合是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，也称为辅因子与底物的“超化学计量比”，在这些基于核酸的酶中DNA切割反应通常需要。此处描述的对内源性顺磁中心（Cu2）的关注为分析涉及几种不同辅因子的混合物铺平了道路。此外，在更复杂的体系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。
