A chemical probe based on the PreQ1 metabolite enables transcriptome-wide mapping of binding sites

Sumirtha Balaratnam,John S Schneekloth Jr,Colleen Connelly,Philip J Homan,James A Kelley,Kamyar Yazdani,Desta Doro Bume,Christopher C Lai,Danny Incarnato,Curran Rhodes,Tomoyuki Numata

doi:10.1038/s41467-021-25973-x

Abstract

The role of metabolite-responsive riboswitches in regulating gene expression in bacteria is well known and makes them useful systems for the study of RNA-small molecule interactions. Here, we study the PreQ1 riboswitch system, assessing sixteen diverse PreQ1-derived probes for their ability to selectively modify the class-I PreQ1 riboswitch aptamer covalently. For the most active probe (11), a diazirine-based photocrosslinking analog of PreQ1, X-ray crystallography and gel-based competition assays demonstrated the mode of binding of the ligand to the aptamer, and functional assays demonstrated that the probe retains activity against the full riboswitch. Transcriptome-wide mapping using Chem-CLIP revealed a highly selective interaction between the bacterial aptamer and the probe. In addition, a small number of RNA targets in endogenous human transcripts were found to bind specifically to 11, providing evidence for candidate PreQ1 aptamers in human RNA. This work demonstrates a stark influence of linker chemistry and structure on the ability of molecules to crosslink RNA, reveals that the PreQ1 aptamer/ligand pair are broadly useful for chemical biology applications, and provides insights into how PreQ1, which is similar in structure to guanine, interacts with human RNAs.

Highlights

The role of metabolite-responsive riboswitches in regulating gene expression in bacteria is well known and makes them useful systems for the study of RNA-small molecule interactions
The study of PreQ1 in human transcriptomes has mostly been limited to covalent modification of tRNAs by queuosine[10]
A broad assessment of PreQ1 binding in human transcriptomes would clarify the selectivity of the PreQ1 aptamer interaction and enable an unbiased examination of metabolite binding to human RNAs

Summary

Introduction

The role of metabolite-responsive riboswitches in regulating gene expression in bacteria is well known and makes them useful systems for the study of RNA-small molecule interactions. This work demonstrates a stark influence of linker chemistry and structure on the ability of molecules to crosslink RNA, reveals that the PreQ1 aptamer/ligand pair are broadly useful for chemical biology applications, and provides insights into how PreQ1, which is similar in structure to guanine, interacts with human RNAs. Riboswitches are naturally occurring RNA sequences that influence bacterial gene expression by binding directly to small molecules[1,2,3]. A broad assessment of PreQ1 binding in human transcriptomes would clarify the selectivity of the PreQ1 aptamer interaction and enable an unbiased examination of metabolite binding to human RNAs. Reactive molecules that covalently modify RNAs have played a substantial role in understanding RNA biology by probing structure[14,15], controlling gene expression[16], imaging RNAs17,18, tagging RNAs with functional handles, and demonstrating target engagement for RNA-binding small molecules[19,20,21,22]. Understanding the chemical features that impact RNA crosslinking efficiency would influence probe design, as well as inform interpretation of results from experiments that use these probes

Methods

Results

Conclusion