英国MRC分子生物学实验室Holliger Philipp团队报道了一个双残基初链空间栅控制2′-O-甲基-和2′-O-(2-甲氧基乙基)- RNA的合成。相关研究成果发表在2022年10月13日出版的《自然—化学》。

位阻排斥是酶底物特异性的关键因素，包括聚合酶。这种底物特异性限制了2′-修饰核酸的酶促合成，这在基于核酸的药物开发中很有意义。

该文中，研究人员描述了在古细菌DNA聚合酶中发现的一个双残基、新生链、空间位阻“门”。研究人员表明，在前面描述的RNA聚合酶活性的背景下，降低空间位阻的门工程开启了2′-修饰RNA低聚物的合成，特别是定义和随机序列2′-O-甲基-RNA（2′OMe RNA）和2′-O-（2-甲氧乙基）-RNA（MOE-RNA）低聚物高达750吨的有效合成。这使得发现了完全由2′OMe-RNA（2′OMezymes）组成的RNA内切酶催化剂，用于癌基因KRAS（G12D）和β-catenin CTNNB1（S33Y）mRNAs的等位基因特异性切割，并制备了对血管内皮生长因子具有高亲和力的混合2′OMe-/MOE-RNA适配体。

研究结果为这些2′-修饰的RNA(几种核准核酸疗法中的应用)在酶合成，以及定向转化和纳米技术的更广泛探索开辟了道路。

附：英文原文

Title: A two-residue nascent-strand steric gate controls synthesis of 2′-O-methyl- and 2′-O-(2-methoxyethyl)-RNA

Author: Freund, Niklas, Taylor, Alexander I., Arangundy-Franklin, Sebastian, Subramanian, Nithya, Peak-Chew, Sew-Yeu, Whitaker, Amy M., Freudenthal, Bret D., Abramov, Mikhail, Herdewijn, Piet, Holliger, Philipp

Issue&Volume: 2022-10-13

Abstract: Steric exclusion is a key element of enzyme substrate specificity, including in polymerases. Such substrate specificity restricts the enzymatic synthesis of 2′-modified nucleic acids, which are of interest in nucleic-acid-based drug development. Here we describe the discovery of a two-residue, nascent-strand, steric control ‘gate’ in an archaeal DNA polymerase. We show that engineering of the gate to reduce steric bulk in the context of a previously described RNA polymerase activity unlocks the synthesis of 2′-modified RNA oligomers, specifically the efficient synthesis of both defined and random-sequence 2′-O-methyl-RNA (2′OMe-RNA) and 2′-O-(2-methoxyethyl)-RNA (MOE-RNA) oligomers up to 750nt. This enabled the discovery of RNA endonuclease catalysts entirely composed of 2′OMe-RNA (2′OMezymes) for the allele-specific cleavage of oncogenic KRAS (G12D) and β-catenin CTNNB1 (S33Y) mRNAs, and the elaboration of mixed 2′OMe-/MOE-RNA aptamers with high affinity for vascular endothelial growth factor. Our results open up these 2′-modified RNAs—used in several approved nucleic acid therapeutics—for enzymatic synthesis and a wider exploration in directed evolution and nanotechnology.

DOI: 10.1038/s41557-022-01050-8

Source: https://www.nature.com/articles/s41557-022-01050-8