整合固态纳米孔和生物膜纳米测序技术研究活体细胞RNA G-四链体形成及RNA-G4解旋酶的分子机理

G-四链体(G4)是由富含重复串联的鸟嘌呤碱基(G)的DNA或RNA链折叠形成的特殊的核酸二级结构。相比DNA-G4，RNA-G4更稳定地存在于活细胞内，并在调节mRNA的转录、修饰及蛋白表达调控上起重要作用。大量研究表明，RNA-G4与个体发育，人类重大疾病如癌症和神经疾病的发生发展密切相关，研究RNA-G4在细胞内形成的调控机理既具有重大的基础生物学意义，又对人类健康及基础医学发展起重要作用。本项目聚焦于世界科技前沿的RNA-G4的热点研究、瞄准有重大争议的学术难点，独辟蹊径, 开发固态纳米孔传感器并将其和3代纳米孔测序仪整合，直接检测来自活体细胞的RNA，实现高通量实时观察鉴定RNA-G4的形成及种类。阐明RNA-G4在活体细胞内形成的条件和机理。鉴定不同RNA-G4解旋酶特定的生理底物，结合单分子操纵技术及结构生物学等手段在分子水平上揭示RNA-G4解旋酶的工作机理。

The G-quadruplex (G4) is a special nucleic acid secondary structure formed by folding DNA or RNA strands that are rich in tandem guanine bases (G). Compared with DNA-G4, RNA-G4 is more stable in living cells and plays an important role in regulating mRNA transcription, modification, and protein expression regulation. A large number of studies have shown that RNA-G4 is closely related to individual development, the occurrence, and the development of major human diseases such as cancer and neurological diseases. Therefore, the study of the regulatory mechanism of RNA-G4 formation in cells has significant basic biological significance and for human health. This project focuses on the hotspots of RNA-G4 research and aims at the major controversial academic difficulties to develop a unique approach of a solid-state nanopore sensor and integrates it with a third-generation nanopore sequencer to directly detect RNA-G4 from living cells with high-throughput and real-time to identify the formation and types of RNA-G4. We are therefore able to clarify the conditions and mechanism of RNA-G4 formation in living cells; to identify specific physiological substrates of different RNA-G4 helicases. Finally, by combining single-molecule manipulation techniques and structural biology to reveal the working mechanism of RNA-G4 helicases at the molecular level.