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通过热稳定的T7RNA聚合酶变体作为催化剂有效合成circRNA

Wei He^1,2, Xinya Zhang², Yangxiaoyu Zou²

¹College of Biology and the Environment, Nanjing Forestry University, Nanjing, China.

Frontiers in bioengineering and biotechnology

|April 24, 2024

概括

一种新的多位元突变T7RNA聚合酶使循环RNAs (circRNAs) 的单合成成为可能. 这种精简的方法实现了高效率,并减少了不必要的副产品,简化了潜在疾病治疗的circRNA生产.

科学领域:

生物化学生物化学
分子生物学分子生物学
在RNA治疗方面,RNA疗法.

背景情况:

循环RNAs (circRNAs) 是具有稳定结构的非编码RNA,显示出治疗复杂疾病的前景.
目前用于circRNA合成的方法,像PIE方法一样,由于酶限制,通常需要多个步骤和特定的温度条件.

研究的目的:

开发一个简化的一步反应,用于circRNA合成.
克服野生型T7RNA聚合酶的热不稳定性,以改善circRNA的产生.

主要方法:

设计了一种具有增强热稳定的多位元突变T7RNA聚合酶 (S430P,N433T,S633P,F849I,F880Y,G788A).
使用共识和折叠自由能量计算来选择热点,以简化合成.
使用热稳定突变物实施了单转录和循环反应.

主要成果:

改造的突变T7RNA聚合酶在45°C下保持稳定的活性超过一个小时.
实现了单cirRNA合成,其效率与传统的两步方法相美.
证明高循环率超过95%并减少了免疫刺激性dsRNA副产品的产生.

结论:

一种热稳定的T7RNA聚合酶突变体促进了简化和高效的单circRNA合成.
这种改进的方法为研究和治疗应用生产circRNA提供了更简单的方法.
减少dsRNA副产品可以提高合成的circRNA的安全性.

关键词:

环RNA 环RNA 是一个环RNA.循环化循环化突变是发生在突变中的突变.综合合成是一种合成.热稳定性热稳定性转录转录是一种转录.

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