Ribozyme‐Catalyzed Site‐Specific Labeling of RNA Using <i>O</i> ⁶ ‐alkylguanine SNAP‐Tag Substrates

Abstract Site‐specific modification of RNAs with functional handles enables studies of RNA structure, fate, function, and interactions. Ribozymes provide an elegant way to covalently modify RNA of interest (ROI). Here, we report that the methyltransferase ribozyme MTR1 can be employed as a versatile tool for RNA modification and labeling. Using O 6 ‐alkylguanine cofactors, designed in analogy to SNAP‐tag substrates for protein labeling, MTR1 installs various bioorthogonal functional groups at N 1 of a specific adenosine in the RNA target. In this application of ribozyme‐catalyzed RNA labeling, MTR1 is now called SNAPR. In contrast to the self‐labeling SNAP‐tag, which is appended to the protein of interest, SNAPR is a truly intermolecular RNA catalyst (active in trans). SNAPR assembles with the ROI to the active ribozyme, allowing for the transfer of clickable tags, such as azide and alkyne moieties, as well as photolabile groups or cross‐linkers from the guanine cofactor to the ROI. Moreover, we demonstrate a two‐step approach to attach labels at N 6 of the target adenosine: first, SNAPR generates N 1 A‐modified RNA, followed by preparative Dimroth rearrangement to produce N 6 A‐modified RNA. We demonstrate this strategy with p ‐azidobenzyl groups as photocrosslinker to generate covalent RNA–protein conjugates. Overall, this work expands the toolbox for site‐specific RNA modification.