解析T7 RNA聚合酶:从构效关系到dsRNA挑战与生物技术应用
CSTR:
作者:
作者单位:

1)中国药科大学工学院,南京 211198;2)苏州晶睿生物科技有限公司,苏州 215000

作者简介:

通讯作者:

中图分类号:

基金项目:

国家生物药技术创新中心揭榜挂帅项目(NCTIB2022HS03002)和江苏省自然科学基金(BK20221255)资助。


Analysis of T7 RNA Polymerase: From Structure-function Relationship to dsRNA Challenge and Biotechnological Applications
Author:
Affiliation:

1)School of Engineering, China Pharmaceutical University, Nanjing 211198, China;2)GeneVide Biotechnology Co., Ltd, Suzhou 215000, China

Fund Project:

This work was supported by grants from “Open Competition to Select the Best Candidates” Key Technology Program for Nucleic Acid Drugs of NCTIB (2022HS03022) and the Natural Science Foundation of Jiangsu Province (BK20221255).

  • 摘要
  • |
  • 图/表
  • |
  • 访问统计
  • |
  • 参考文献
  • |
  • 相似文献
  • |
  • 引证文献
  • |
  • 资源附件
  • |
  • 文章评论
    摘要:

    T7 RNA聚合酶(T7 RNA polymerase,T7 RNAP)凭借其独特的结构特性成为研究RNA合成机制的重要模型。本文系统解析了其标志性的“右手”结构框架,并通过整合结构的动态变化和动力学分析,阐述了完整的T7 RNAP转录过程,构建了从静态结构解析到动态过程的完整框架。T7 RNAP在催化过程中会产生副产物双链RNA(dsRNA),其存在不仅会降低mRNA产物的纯度,还会引发自身非特异性免疫反应,从而限制T7 RNAP在生物技术和医学领域的应用。文中详细探讨了dsRNA的形成机理,并对规避dsRNA产生的不同思路及当前研究进展进行了阐述。通过梳理近期研究成果,本文系统归纳了通过半理性设计改造的T7 RNAP突变及突变效果,如活性和热稳定性的提升、底物和启动子特异性的改变、转录效率改善等,清晰展现该领域的技术突破路径。此外,T7 RNAP在基因编辑及基因工程、检测诊断及信号转导、mRNA疫苗等领域得到快速发展和广泛应用。本文综述了T7 RNAP的结构功能、dsRNA形成机理及规避策略,同时探讨了T7 RNAP工程优化与功能拓展,并对目前亟待解决的问题进行了阐述,讨论了当前T7 RNAP实际应用中的主要问题并对未来研究的可能方向进行了展望,旨在为T7 RNAP的研发与应用提供新的见解,促进相关领域的思考与发展。

    Abstract:

    T7 RNA polymerase (T7 RNAP) is one of the simplest known RNA polymerases. Its unique structural features make it a critical model for studying the mechanisms of RNA synthesis. This review systematically examines the static crystal structure of T7 RNAP, beginning with an in-depth examination of its characteristic “thumb”, “palm”, and “finger” domains, which form the classic “right-hand-like” architecture. By detailing these structural elements, this review establishes a foundation for understanding the overall organization of T7 RNAP. This review systematically maps the functional roles of secondary structural elements and their subdomains in transcriptional catalysis, progressively elucidating the fundamental relationships between structure and function. Further, the intrinsic flexibility of T7 RNAP and its applications in research are also discussed. Additionally, the review presents the structural diagrams of the enzyme at different stages of the transcription process, and through these diagrams, it provides a detailed description of the complete transcription process of T7 RNAP. By integrating structural dynamics and kinetics analyses, the review constructs a comprehensive framework that bridges static structure to dynamic processes. Despite its advantages, T7 RNAP has a notable limitation: it generates double-stranded RNA (dsRNA) as a byproduct. The presence of dsRNA not only compromises the purity of mRNA products but also elicits nonspecific immune responses, which pose significant challenges for biotechnological and therapeutic applications. The review provides a detailed exploration of the mechanisms underlying dsRNA formation during T7 RNAP catalysis, reviews current strategies to mitigate this issue, and highlights recent progress in the field. A key focus is the semi-rational design of T7 RNAP mutants engineered to minimize dsRNA generation and enhance catalytic performance. Beyond its role in transcription, T7 RNAP exhibits rapid development and extensive application in fields, including gene editing, biosensing, and mRNA vaccines. This review systematically examines the structure-function relationships of T7 RNAP, elucidates the mechanisms of dsRNA formation, and discusses engineering strategies to optimize its performance. It further explores the engineering optimization and functional expansion of T7 RNAP. Furthermore, this review also addresses the pressing issues that currently need resolution, discusses the major challenges in the practical application of T7 RNAP, and provides an outlook on potential future research directions. In summary, this review provides a comprehensive analysis of T7 RNAP, ranging from its structural architecture to cutting-edge applications. We systematically examine: (1) the characteristic right-hand domains (thumb, palm, fingers) that define its minimalistic structure; (2) the structure-function relationships underlying transcriptional catalysis; and (3) the dynamic transitions during the complete transcription cycle. While highlighting T7 RNAP’s versatility in gene editing, biosensing, and mRNA vaccine production, we critically address its major limitation—dsRNA byproduct formation—and evaluate engineering solutions including semi-rationally designed mutants. By synthesizing current knowledge and identifying key challenges, this work aims to provide novel insights for the development and application of T7 RNAP and to foster further thought and progress in related fields.

    参考文献
    相似文献
    引证文献
引用本文

宁苇辰,华雨,尤慧玲,李秋实,吴尧,刘云龙,胡振新.解析T7 RNA聚合酶:从构效关系到dsRNA挑战与生物技术应用[J].生物化学与生物物理进展,2025,52(9):2280-2294

复制
相关视频

分享
文章指标
  • 点击次数:
  • 下载次数:
  • HTML阅读次数:
  • 引用次数:
历史
  • 收稿日期:2025-03-17
  • 最后修改日期:2025-09-13
  • 录用日期:2025-07-08
  • 在线发布日期: 2025-07-10
  • 出版日期:
文章二维码
关闭