上海交通大学刘昱团队报道了在特定位置修饰大RNA的DNA聚合酶工程。相关研究成果发表在2025年1月13日出版的《自然—化学》。

在特定位置进行精确修饰的大RNA合成，对基础研究和治疗应用都有很高的需求，但有效的方法有限。工程DNA聚合酶最近已成为RNA标记的有吸引力的工具，与传统的RNA聚合酶相比具有明显的优势。

该文中，研究人员通过半理性设计，设计了一种DNA聚合酶变体，并将其用于将各种修饰（包括碱基修饰、2′-核糖修饰和骨架修饰）精确地结合到RNA中的所需位置。在大多数修饰情况下，其效率超过85%，证明了将2′-O-甲基、硫代磷酸酯、N4乙酰胞苷和荧光团，引入eGFP和萤火虫荧光素酶信使RNA的特定位点是成功的。

N4乙酰胞苷、2′-O-甲基和/或硫代磷酸酯的mRNA产品，已经证明了增强稳定性和影响蛋白质生产的能力。这种方法为RNA的全面功能化提供了一种有前景的工具，其能够引入大量的修饰，而不管RNA的长度和序列如何。

附：英文原文

Title: Engineering a DNA polymerase for modifying large RNA at specific positions

Author: Chen, Dian, Han, Zhanghui, Liang, Xiaoge, Liu, Yu

Issue&Volume: 2025-01-13

Abstract: The synthesis of large RNA with precise modifications at specific positions is in high demand for both basic research and therapeutic applications, but efficient methods are limited. Engineered DNA polymerases have recently emerged as attractive tools for RNA labelling, offering distinct advantages over conventional RNA polymerases. Here, through semi-rational designs, we engineered a DNA polymerase variant and used it to precisely incorporate a diverse range of modifications, including base modifications, 2′-ribose modifications and backbone modifications, into desired positions within RNA. We achieved efficiencies exceeding 85% in the majority of modification cases, demonstrating success in introducing 2′-O-methyl, phosphorothioate, N4-acetylcytidine and a fluorophore to specific sites in eGFP and Firefly luciferase messenger RNA. Our mRNA products with N4-acetylcytidine, 2′-O-methyl and/or phosphorothioate have demonstrated the ability to enhance stability and affect protein production. This method presents a promising tool for the comprehensive functionalization of RNA, enabling the introduction of plentiful modifications irrespective of RNA lengths and sequences.

DOI: 10.1038/s41557-024-01707-6

Source: https://www.nature.com/articles/s41557-024-01707-6