小柯机器人

美国加州大学Stephen N. Floor团队近期取得重要工作进展，他们研究提出使用工程化的RNA脱氨酶进行RNA分子记录的方法。相关研究成果2023年10月19日在线发表于《自然—方法学》杂志上。

据介绍，RNA脱氨酶是碱基编辑和RNA分子记录的强大工具。然而，由于RNA结构和序列依赖性，目前可用的RNA分子记录器如TRIBE、DART或STAMP中使用的酶具有局限性。

研究人员为RNA分子记录器的定向演化设计了一个平台，他们开发出了一种具有高活性、低偏倚和低背景的RNA A-to-I脱氨酶（一种RNA腺苷碱基编辑器，rABE）。使用rABE，研究人员提出了REMORA（腺苷中RNA编码的分子记录），其中rABE的脱氨作用记录了RNA-蛋白质相互作用的分子记录。通过将rABE与C-to-U脱氨酶APOBEC1和长读RNA测序相结合，研究人员测量了两种RNA结合蛋白与单个信使RNA（mRNA）的结合。哺乳动物Pumilio蛋白PUM1和PUM2的正交RNA分子记录显示，PUM1与PUM2竞争细胞中的一个子集位点。

此外，研究人员鉴定了由结合位点远端的同种型变化驱动的转录物同种型特异性RNA-蛋白质相互作用。基因编码的RNA脱氨酶rABE能够单分子鉴定具有细胞类型特异性的RNA-蛋白质相互作用。

附：英文原文

Title: RNA molecular recording with an engineered RNA deaminase

Author: Lin, Yizhu, Kwok, Samentha, Hein, Abigail E., Thai, Bao Quoc, Alabi, Yewande, Ostrowski, Megan S., Wu, Ke, Floor, Stephen N.

Issue&Volume: 2023-10-19

Abstract: RNA deaminases are powerful tools for base editing and RNA molecular recording. However, the enzymes used in currently available RNA molecular recorders such as TRIBE, DART or STAMP have limitations due to RNA structure and sequence dependence. We designed a platform for directed evolution of RNA molecular recorders. We engineered an RNA A-to-I deaminase (an RNA adenosine base editor, rABE) that has high activity, low bias and low background. Using rABE, we present REMORA (RNA-encoded molecular recording in adenosines), wherein deamination by rABE writes a molecular record of RNA–protein interactions. By combining rABE with the C-to-U deaminase APOBEC1 and long-read RNA sequencing, we measured binding by two RNA-binding proteins on single messenger RNAs. Orthogonal RNA molecular recording of mammalian Pumilio proteins PUM1 and PUM2 shows that PUM1 competes with PUM2 for a subset of sites in cells. Furthermore, we identify transcript isoform-specific RNA–protein interactions driven by isoform changes distal to the binding site. The genetically encodable RNA deaminase rABE enables single-molecule identification of RNA–protein interactions with cell type specificity.

DOI: 10.1038/s41592-023-02046-z

Source: https://www.nature.com/articles/s41592-023-02046-z

Nature Methods：《自然—方法学》，创刊于2004年。隶属于施普林格·自然出版集团，最新IF：47.99
官方网址：https://www.nature.com/nmeth/
投稿链接：https://mts-nmeth.nature.com/cgi-bin/main.plex