基于多重单分子技术研究nsp13解旋酶调控新冠病毒转录复制过程的分子机制

新冠病毒SARS-CoV-2造成了全球大流行性疫情，研究复制转录与致病机制，从而有效抑制病毒的传播及感染力，成为当前生物医学领域的关键问题。结构生物学研究了SARS-CoV-2的Holo-RdRp聚合全酶和nsp13解旋酶的精细结构，为病毒防御提供了重要的基础。但是二者的动态作用机制研究还相对薄弱。本项目拟采用多重单分子技术：① 基于磁镊技术设计单分子移位实验，追踪nsp13与聚合全酶互作的动态过程，揭示nsp13调控RTC复合物的分子机制；② 采用单分子荧光耦合磁镊技术，利用荧光探针标记的聚合全酶、nsp13，实时追踪RTC复合物的组装过程，研究移位复合物的构成组分；③ 检测瑞德西韦药物是否延迟或终止转录复制，研究瑞德西韦是否影响nsp13移位的功能。研究nsp13在新冠病毒转录复制中的动态调控机制，能够为研发靶向Holo-RdRp和nsp13抗病毒药物以及疫苗研究提供重要的理论依据。

The new coronavirus SARS-CoV-2 has caused a global pandemic COVID-19. The investigation of replication, transcription and infection mechanisms, which helps effectively inhibit the spread and infectivity of the virus, has become a key issue in the current biomedical field. Structural studies have revealed the fine structure of Holo-RdRp polymerase and nsp13 helicase of SARS-CoV-2, providing an important basis for virus defense. However, the research on the dynamic interaction between Holo-RdRp and nsp13 is still relatively weak. This proposal intends to employ multiple single-molecule techniques: ① using biotin-labeled Holo-RdRp to design a single-molecule translocation assay based on single-molecule magnetic tweezers, to track the dynamic interaction between Holo-RdRp and nsp13 in real time. This proposal plans to reveal the molecular mechanism regarding how nsp13 helicase regulates RTC complex. ② It is also planned to adopt the tandem technology of single-molecule fluorescence-coupled magnetic tweezers, using photostable fluorescent probes to label Holo-RdRp and nsp13 helicase, to track the assembly process of the RTC complex in real time, to detect the composition of the translocation complex. ③ It is essential to detect whether Remdesivir drug can delay or slow down transcription process of SARS-CoV-2 and to reveal whether Remdesivir may influence of nsp13 translocation on the RNA chain. In summary, our investigation of the dynamic regulation mechanism of nsp13 helicase in SARS-CoV-2 transcription and replication can provide an important theoretical evidence for the development of antiviral drugs targeting Holo-RdRp and nsp13 and vaccine research.