A chemical probe based on the PreQ 1 metabolite enables transcriptome-wide mapping of binding sites

• Published in: Nature communications 2021-10, Vol.12 (1), p.5856
• Main Authors: Balaratnam, Sumirtha, Rhodes, Curran, Bume, Desta Doro, Connelly, Colleen, Lai, Christopher C, Kelley, James A, Yazdani, Kamyar, Homan, Philip J, Incarnato, Danny, Numata, Tomoyuki, Schneekloth, Jr, John S
• Format: Article
• Language: English
• Subjects: Binding Sites; Crystallography, X-Ray; Humans; Ligands; Pyrimidinones - chemistry; Pyrimidinones - metabolism; Pyrroles - chemistry; Pyrroles - metabolism; Riboswitch; RNA, Bacterial - genetics; Transcriptome
• ISSN: 2041-1723
• DOI: 10.1038/s41467-021-25973-x

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