生物学杂志

生物学杂志 ›› 2026, Vol. 43 ›› Issue (2): 97-.doi: 10.3969/j.issn.2095-1736.2026.02.097

• 技术方法 • 上一篇    下一篇

新型特异性探针在mRNA加帽率检测中的应用

王珍珍1， 陈　婷1， 张云龙1， 陆昌瑞1， 张贵霖2   

  1. 1. 东华大学 生物与医学工程学院， 上海 201620； 2. 上海麦野生物科技有限公司， 上海 201508
  • 出版日期:2026-04-18 发布日期:2026-04-23
  • 通讯作者: 张贵霖，监事，研究方向为核酸药物，E-mail：zy@meyesbio.com；陆昌瑞，博士，教授，研究方向为RNA的结构和功能，E-mail：crlu@dhu.edu.cn；张贵霖和陆昌瑞为共同通信作者
  • 作者简介:王珍珍，硕士研究生，研究方向为mRNA疫苗研发，E-mail：wzz2018138107@163.com
  • 基金资助:
    上海市科学技术委员会国际合作计划项目(19410711000)

Application of novel specific probes for mRNA capping rate detection

WANG Zhenzhen1, CHEN Ting1, ZHANG Yunlong1, LU Changrui1, ZHANG Guilin2   

  1. 1. School of Biological and Medical Engineering, Donghua University, Shanghai 201620, China;
    2. Shanghai Meyesbio Technology Co. Ltd., Shanghai 201508, China
  • Online:2026-04-18 Published:2026-04-23

摘要： mRNA疫苗凭借高效免疫保护及灵活靶向设计优势，迅速成为全球公共卫生与精准医疗领域的革命性技术。加帽率检测是mRNA技术从实验室走向工业化应用的质量基石，其精度与效率直接决定药物的安全性、有效性及生产成本。现有加帽率检测方法在准确性、效率、成本及规模化应用方面仍有不足。为探索mRNA疫苗质量控制的新策略，本研究通过筛选新型特异性探针及简化下游纯化方式对现有核糖核酸酶H（RNase H）分析方法进行完善与创新。共设计17个具有不同长度和起始位点的探针，通过RNase H酶切和液相色谱—质谱（LC-MS）法进行特异性筛选。随后，利用筛选出的探针与底物mRNA退火杂交，采用RNase H切割杂交链中的mRNA。切割产物经硅胶柱分离，小片段进一步通过LC-MS分析计算得出加帽率。结果显示，筛选到3个不同长度探针，能特异性诱导RNase H在－2位点进行高效切割，切割频率大于99%。硅胶柱纯化技术有效实现了对短片段的分离，成功剔除了长片段对实验结果的潜在干扰。该方法应用于mRNA加帽率检测，结果稳定在97%左右。新型特异性探针以其高效、精准及高性价比，适用于mRNA疫苗加帽率质控检测。

关键词: mRNA疫苗, 质量控制, 加帽率检测, RNase H, LC-MS

Abstract: The mRNA vaccine platform has emerged as a revolutionary technology in global public health and precision medicine, driven by its high immunoprotective efficacy, and flexible target design. As a critical quality attribute in industrial-scale mRNA production, capping efficiency directly impacts drug safety, efficacy, and manufacturing costs. However, current analytical methods suffer from limitations in accuracy, throughput, cost-effectiveness, and scalability. To address these challenges, this study innovatively optimized RNase H-based analytical strategies through rational probe design and streamlined purification processes. Seventeen sequence-specific probes with varied lengths and hybridization sites were systematically evaluated through RNase H digestion followed by LC-MS characterization. Three optimized probes demonstrated precise RNase H-mediated cleavage at the －2 position with over 99% efficiency. Subsequent silica-based purification effectively eliminated interference from uncut mRNA fragments, achieving consistent capping rates of 97% across multiple batches. This refined methodology establishes a robust, cost-effective platform for capping efficiency assessment, significantly advancing quality control paradigms for mRNA vaccine development.

Key words: mRNA vaccine, quality control, cap rate detection, RNase H, LC-MS

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