阳离子型钌金属肽的构建及对分子间三螺旋RNA的结构调控研究

Intermolecular triple-helical RNA (also called RNA triplexes) present a variety of potential applications in many biological processes such as transcriptional regulation, RNA processing, chromatin modification and inhibition of oncogenic miRNA precursor producing cancer genes. Due to the Hoogsteen base pairing, however, RNA triplexes are thermodynamically less stable than the corresponding Watson-Crick base paired duplex strand, and the formation processes of triple-helical structures are also very slow in comparison with those of the corresponding template duplexes. The two factors hinder the practical applications of RNA triplexes at present and need to be resolved urgently. To achieve the rapid formation, balance, and stability of a triple helix, how to lower the interchain electrostatic repulsion and enhance the binding force between the double helical and a third strand is a significant and practical topic. This project intends to construct spermine and lysine-rich cationic ruthenium(II) metallopeptides by (1) using polypyridyl ruthenium group as chromophores and basic amino acids forming cationic amino acid residues in peptide side chains, (2) controlling neutral amino acids existence or not in peptide backbones and varying peptide sequence and the fraction of basic amino acid residues and (3) alternating the ring plane, the electron density and the spatial effect of crescent shaped aryls. A rapid formation and stability of a triple helix could be achieved by (1) reducing the electrostatic repulsion between the double helix and the incoming third strand oligonucleotide by selective recognition and binding of basic amino acid residues to the negatively charged phosphates, (2) strong intermolecular interaction involving effective overlap of the π electron cloud of crescent shaped aryls with the base triplets. The implementation of this project will further advances our knowledge on the regulatory mechanisms of forming processes and structural stabilities of intermolecular RNA triplexes by small chemical molecules, which provides new ideas for improving the formation kinetics and stability of RNA triplexes.

分子间三螺旋 RNA在转录调控、RNA转录后加工、染色质修饰、抑制致癌miRNA前体产生癌基因等生物过程具有广泛的应用前景。然而生理条件下其结构慢形成过程和低稳定性是阻碍三螺旋技术实际应用而有待解决的关键问题。本项目拟以多吡啶钌基为生色团、碱性氨基酸构成肽侧链上的阳离子氨基酸残基，控制肽骨架链有无中性氨基酸，调节碱性氨基酸的组分和序列及新月形芳基的环平面、电子密度和空间效应，构建富含碱性赖氨酸或精氨酸的阳离子型钌金属肽。通过优化溶液环境，调节阳离子型钌金属肽侧链上碱性氨基酸残基对磷酸官能团的选择识别与结合、新月形芳基与碱基对间的堆积作用及其他功能基的作用，降低链间静电排斥力，提高三链间的结合力，实现分子间三螺旋RNA的快速形成与平衡及结构稳定。本项目的实施，将加深理解化学小分子对分子间三螺旋RNA形成过程和结构稳定的调控机制及其影响因素，为改善分子间三螺旋RNA结构的形成和稳定提供新思路。

分子间三螺旋RNA在转录调控、RNA转录后加工、染色质修饰等生物过程具有广泛应用前景，然而生理条件下其结构稳定性低是阻碍三螺旋技术面向实际应用而有待解决的关键问题之一。本项目通过以多吡啶钌基为生色团、碱性氨基酸构成肽侧链上的阳离子氨基酸残基，控制肽骨架链有无中性氨基酸，调节碱性氨基酸的组分和序列，合成了系列富含碱性赖氨酸或精氨酸的阳离子型钌金属肽，并应用光谱学方法与核酸热变性技术探讨了其对三螺旋RNA的结合与稳定作用。研究结果表明，调节阳离子氨基酸残基的种类、数目与分布，可调控阳离子型钌金属肽的碱性氨基酸残基对磷酸官能团的选择识别与结合；优化阳离子型钌金属肽的浓度以及溶液的离子浓度和pH等因素，可实现生理条件下阳离子型钌金属肽对分子间三螺旋RNA结构稳定的调控。在正电荷与氨基酸残基数目相同的条件下，含精氨酸残基的阳离子型钌金属肽较含赖氨酸残基的阳离子型钌金属肽表现出对三螺旋第三链更强的稳定作用：含精氨酸残基的阳离子型钌金属肽倾向于稳定三螺旋的第三链而不是模板链，而含赖氨酸残基的阳离子型钌金属肽通常优先稳定三螺旋的模板链而不是第三链。另外，显著不同于插入剂钌多吡啶配合物，辅助配体的疏水性与平面大小通常对阳离子型钌金属肽结合与稳定三螺旋RNA不具明显的影响，但阳离子型钌金属肽电荷数的改变会对其结合与稳定三螺旋产生非常明显的影响。对于以静电作用与三螺旋RNA相互结合的钌金属肽，钌金属肽的浓度对其稳定三螺旋具有非常重要的影响，通常钌金属肽在低浓度时对三螺旋RNA具有非常强的稳定作用，然而其稳定三螺旋的作用随着钌金属肽浓度的增大而减弱。与钌金属肽肽链中的正电荷的分布相比较，碱性氨基酸的种类与数目是影响其结合与稳定三螺旋RNA的关键因素。本项目的实施，将加深理解小分子对分子间三螺旋RNA结构稳定的调控机制及其影响因素，为改善分子间三螺旋RNA的结构稳定提供新思路。

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