A chemical probe based on the PreQ 1 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 PreQ 1 riboswitch system, assessing sixteen diverse PreQ 1 -derived probes for their ability to selectively modify the class-I PreQ 1 riboswitch aptamer covalently. For the most active probe ( 11 ), a diazirine-based photocrosslinking analog of PreQ 1 , 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 PreQ 1 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 PreQ 1 aptamer/ligand pair are broadly useful for chemical biology applications, and provides insights into how PreQ 1 , which is similar in structure to guanine, interacts with human RNAs.