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