摘要
目的为了阐明病毒复制转录中的动力学特征,建立病毒体外复制转录模型,利用单分子磁镊技术探究SARS-CoV-2 RNA依赖的RNA聚合酶(RNA-dependent RNA polymerase,RdRp)体外复制转录的动态过程。方法以DNA片段为研究对象进行单分子磁镊系统的力场校正,随后构建RNA片段探究RdRp体外复制转录动力学。结果力场校正结果符合蠕虫链模型(worm-like chain model,WLC)。在30 pN力场下ssRNA链比dsRNA链长约0.3μm且能稳定延伸,RdRp的延伸平均合成速率3.27 nt/s,RdRp的持续合成活性为平均886 nt。结论实现了对单分子磁镊力场校正,通过实时监测RdRp体外复制转录起始、延伸和终止等阶段动力学表征,建立RdRp的体外复制转录模型。研究结果为进一步利用单分子磁镊技术研究病毒RdRp在不同体外环境下复制、转录和回溯等生理过程的动力学提供基础,同时为在体外研究不同药物对病毒复制转录过程的影响构建单分子磁镊模型。
Objective To elucidate the kinetic characteristics of viral replication and transcription,an in vitro model of viral replication and transcription was established.Utilizing single-molecule magnetic tweezers technology,the dynamic process of SARS-CoV-2 RNA-dependent RNA polymerase(RdRp)during in vitro replication and transcription was investigated.Methods The force field of the single-molecule magnetic tweezer system was corrected using DNA fragments,followed by the construction of RNA fragments to explore the kinetics of RdRp replication and transcription in vitro.Results The force field calibration results were consistent with the worm-like chain model(WLC).The ssRNA strand was found to be approximately 0.3μm longer than the dsRNA strand and could be stably extended under a 30 pN force field.The average synthesis rate of RdRp extension was determined to be 3.27 nt/s,with an average processivity of 886 nt.Conclusions By implementing force calibration in single-molecule magnetic tweezers,the real-time tracking of RdRp kinetics during the full-cycle replication-transcription process(initiation,elongation,and termination)in vitro was achieved,thereby constructing a mechanistic model of RdRp-driven nucleic acid synthesis.This study provides a basis for further investigating the kinetics of viral RdRp in physiological processes including replication,transcription,and backtracking under varied in vitro environments using single-molecule magnetic tweezers,and establishes a single-molecule manipulation framework for evaluating the effects of therapeutic compounds on viral replication-transcription processes in vitro.
作者
郭芊宏
陈晓敏
郑志勤
樊军
GUO Qianhong;CHEN Xiaomin;ZHENG Zhiqin;FAN Jun(Southwest University of Science and Technology,School of Life Science and Engineering,Mianyang 621000,Sichuan,China;University of Electronic Science and Technology of China,Institute of Fundamental and Frontier Sciences,Chendu 610000,China;School of Biological of Engineering,Sichuan University of Science&Engineering,Yibin 644000,Sichuan,China;NHC Key Laboratory of Nuclear Technology Medical Transformation,Mianyang Central Hospital,Sichuan University of Science&Engineering,Mianyang 621010,Sichuan,China)
出处
《医用生物力学》
北大核心
2025年第4期895-901,共7页
Journal of Medical Biomechanics
基金
国家自然科学基金项目(32101049)。
关键词
单分子磁镊
蠕虫链模型
RNA依赖的RNA聚合酶
复制转录
single-molecule magnetic tweezers
worm-like chain model
RNA-dependent RNA polymerase
replicative transcription
作者简介
共同通信作者:郑志勤,副教授,E-mail:zhengzhiqin@swust.edu.cn;共同通信作者:樊军,教授,E-mail:fan.jun@uestc.edu.cn。
