RNA–protein interactions play numerous roles in cellular function and disease. Here we describe RNA–protein interaction detection (RaPID), which uses proximity-dependent protein labeling, based on the BirA* biotin ligase, to rapidly identify the proteins that bind RNA sequences of interest in living cells. RaPID displays utility in multiple applications, including in evaluating protein binding to mutant RNA motifs in human genetic disorders, in uncovering potential post-transcriptional networks in breast cancer, and in discovering essential host proteins that interact with Zika virus RNA. To improve the BirA*-labeling component of RaPID, moreover, a new mutant BirA* was engineered from Bacillus subtilis, termed BASU, that enables >1,000-fold faster kinetics and >30-fold increased signal-to-noise ratio over the prior standard Escherichia coli BirA*, thereby enabling direct study of RNA–protein interactions in living cells on a timescale as short as 1 min.

RNA与蛋白质的相互作用在细胞功能和疾病中起着许多作用。在这里，我们描述RNA-蛋白质相互作用检测（RaPID），该方法基于BirA *生物素连接酶使用邻近依赖性蛋白质标记，以快速鉴定与活细胞中感兴趣的RNA序列结合的蛋白质。RaPID在多种应用中显示出实用性，包括评估与人类遗传疾病中突变RNA基序的蛋白质结合，发现乳腺癌中潜在的转录后网络以及发现与寨卡病毒RNA相互作用的必需宿主蛋白。此外，为了改善RaPID的BirA *标记成分，从枯草芽孢杆菌（Bacillus subtilis）设计了一个新的突变体BirA *称为BASU，与先前的标准大肠杆菌BirA *相比，可以使动力学加快1000倍以上，信噪比提高30倍以上，从而可以在短时间内直接研究活细胞中RNA-蛋白质相互作用1分钟