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

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.