离子调节G-Quadruplex形成过程与构型可控转化的机制研究

Based on the conformational switches from G-rich aptamer to G-quadruplex, various detecting platforms are developed. However, there are two aspects needed to be carefully reconsidered. If the target is a metal ion, the selected functional aptamer is supposed to undergo a conformational switch to G-quadruplex. Based on the conformational change and the integrated fluorescent or electrochemical probe, the detection is achieved in buffer solution and real samples. The question is that upon increasing the concentration of metal ion, G-quadruplex is quantitatively formed, OR gradually undergone structural transfer from unstable to stable? When the concentration of metal ion is relatively lower and coexisting metal ion is relatively higher, how the coexisting metal ion alters stability and properties of the formed G-quadruplex, and further affects the sensing performance? Misconception remains common. On the other hand, when functional aptamers (through adding more nucleic bases at 3 or 5 terminal) were designed to detect targets, could the added nucleic bases alter the conformation of G-quadruplex, and further affect the binding efficiency with fluorescent or electrochemical probe? Then, how to select the proper functional aptamer to improve the sensing performance? The question is avoided. Thus, the project is supposed to find clues to answer the two mentioned questions. Firstly, it will deeply investigate the transforming process from aptamer into G-quadruplex induced by ions, and its binding efficiency with molecular probes. Meanwhile, coexisting ion mediating and controlling transformation of G-quadruplex will also be explored in detail. The remained misconceptions will be clarified, and the possible approaches will be investigated. Secondly, in order to specifically discriminate the ion-mediated and controlled transformation of formed G-quadruplex, the newly fluorescent or electrochemical probes will be designed and synthesized. Accordingly, the new fluorescent or electrochemical sensors and logic gates will be developed. All the supposed research works will provide a new theory and approach to broaden the application of G-quadruplex in sensing areas.

针对基于富G适体链形成G4结构的检测平台，存在两方面的问题：(1) 随着目标离子浓度的增加，是G4结构形成的数量逐渐增加, 还是构型渐变并趋于稳定？当目标离子浓度较低时，实际体系中的共存离子如何影响G4结构的构型和稳定性，进而改变检测响应性？目前尚存理解性误区；(2)功能化探针适体链（增加适体链3’或5’端的碱基）在特定离子诱导下形成G4结构检测目标物，增加的碱基是否改变G4结构以及G4结构与探针分子的有效键合，进而影响检测响应性？也是一个值得关注的问题。因此，本项目拟深入研究离子诱导探针适体链(含功能化)形成G4结构的过程及其与探针分子的键合，探究离子调节G4结构的可控转化，揭示传感器设计中存在的问题并提出可参考的解决方案；设计并合成能特异识别G4构型可控转化的光/电探针，研发新型光/电传感和逻辑器件。这些研究工作的逐一展开和顺利实施，为拓展G4 结构在传感领域中的应用提供新理论和新方法

本项目旨在研究基于富G适体链形成G4结构检测平台的两个关键问题：(1) 随着目标离子浓度的增加，是G4结构形成的数量逐渐增加, 还是构型渐变并趋于稳定？当目标离子浓度较低时，实际体系中的共存离子如何影响G4结构的构型和稳定性，进而改变检测响应性？(2) 功能化探针适体链（增加适体链3’或5’端的碱基）在特定离子诱导下形成G4结构检测目标物，增加的碱基是否改变G4结构以及G4结构与探针分子的有效键合，进而影响检测 响应性？研究结果显示： (i) 富G适体链识别K+，随着K+浓度的增加，K+-G4结构由单体逐渐发生二聚，并伴随稳定性的逐渐增加，对K+的响应呈现三段线性关系。 (ii) 在Na+体系中识别K+，发现了Na+-G4逐渐向K+-G4转化的过程，惊喜地探测到了Na+-K+-G4中间体；以Na+-K+-G4中间体为探针识别K+，极大地改善了检测响应性；随后，通过在适体链的3’和5’端增加碱基数量和变化碱基种类，在G4结构和冗余互补碱基形成的空腔内可高效地结合荧光小分子，实现了在不同Na+体系中归一化检测K+。 (iii) 富G适体链识别Pb2+时, Na+的存在会显著减低检测Pb2+的性能，究其因是Na+的存在会诱导Pb-G4构型发生反转，并逐渐二聚；基于这一事实，采用目标链DNA与适体链杂化的策略，有效规避了Na+的干扰。 (iv) K+和Pb2+竞争性地与富G适体链作用形成G4结构，K+-G4与Pb2+-G4的结构转化极大地依赖于K+和Pb2+的浓度。有趣的是，当K+浓度大于10 mM时， K+能置换稳定Pb2+-G4结构中的Pb2+并生成稳定K+-G4，这一结果突破了G4结构中K+与Pb2+置换的局限。通过本项目的实施，深入认识了G4结构与离子的依赖性，为拓展G4结构在生物传感，药物递送和抗癌药物的设计提供了有意义的理论指导，也为适体链的筛选提供了有价值的实验指导。

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