工程T7RNA聚合酶通过降低终端转移酶和RNA依赖RNA聚合酶活动来减少dsRNA的形成
Qiongwei Tang1, Sisi Zhu1, Nannan Hu2
1Yeasen Biotechnology (Shanghai) Co., Ltd., China.
The FEBS journal
|March 3, 2025
概括
研究人员设计了T7RNA聚合酶 (RNAP) 来减少双链RNA (dsRNA) 的副产品. 这种工程改善了蛋白质翻译并减轻了免疫应激,提高了体外转录应用的安全性.
科学领域:
- 生物技术是生物技术.
- 分子生物学分子生物学
- 酵素工程是什么? 酶工程是什么
背景情况:
- T7RNA聚合酶 (RNAP) 在体外转录 (IVT) 中至关重要.
- 然而,T7 RNAP产生双链RNA (dsRNA) 副产品,导致免疫应激和安全问题.
- 减少dsRNA对于安全有效的IVT应用是必不可少的.
研究的目的:
- 为了设计具有显著减少dRNA生产的T7RNAP变体.
- 调查dSRNA产生与T7RNAP的终端转移酶和RNA依赖RNAP (RDRP) 活动之间的相关性.
- 评估工程T7RNAP变体对细胞的影响.
主要方法:
- 基于分子信标的光激活滴滴分类 (FADS) 与局部定向突变发生的结合.
- 选的随机图书馆对T7RNAP突变具有减少的dsRNA.
- 进行细胞实验以评估免疫反应和蛋白质翻译.
主要成果:
- 确定了四种T7RNAP突变 (M1,M7,M11,M14) 具有减少dSRNA的产生.
- 组合突变M17在各种条件下显示显著较低的dsRNA水平.
- 突变者在细胞分析中显示了减轻免疫应激和增强蛋白质翻译.
- 观察到dSRNA生产和终端转移酶/RDRP活动之间存在强烈的相关性.
结论:
- 开发了一种用于工程T7 RNAP的新方法.
- 成功选了T7RNAP变体,减少了dsRNA的产生.
- 证明了工程变体在IVT中提高产品完整性和安全性的潜力.
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