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

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.

Targeting RNA structures with small molecules

“…Cellular target validation methods for RNA have been recently developed based on covalent bond formation with 65 , 131 – 133 , or cleavage of, the target 65 . One covalent method, chemical cross-linking and isolation by pull-down (Chem-CLIP; Fig.…”

“…Quantitative PCR with reverse transcription (RT–qPCR) or RNA-seq and subsequent statistical analysis are used to analyse the enrichment of RNA targets in the pulled-down fraction, as compared with the starting lysate. Although the exact protocol may vary depending upon the chemistry of cross-linking 133 and purification modules, the fundamental idea of Chem-CLIP remains unchanged: transformation of dynamic reversible binding to covalent bonds by cross-linking and amplifying the signal by pull-down enrichment. Chem-CLIP experiments are completed side by side with a probe that lacks the RNA-binding module, controlling for nonspecific reaction of the cross-linking module.…”

“…Such data are key to inform lead optimization. Intriguingly, fragment mapping and Chem-CLIP 108 , 133 , 274 , in combination with RNA structure prediction programs such as ScanFold, can provide insight into ligandable cellular RNA structures; biologically silent interactions inform cellular RNAs amenable to targeting by small-molecule-induced degradation.…”

“…Cellular target validation methods for RNA have been recently developed based on covalent bond formation with 65,[131][132][133] , or cleavage of, the target 65 (for example, biotin or an alkyne) 65 . The RNA-binding module drives target engagement, bringing the cross-linking module into proximity to the RNA such that they react either directly (electrophilic in the case of chlorambucil) or upon irradiation (diazirine).…”

Pervasive Transcriptome Interactions of Protein-Targeted Drugs

“…Little is known about the RNA off-target interactions of protein-targeted drugs in vivo, especially for clinically approved drugs. Investigation of small molecule-RNA interactions has been carried out recently via photocrosslinking (diazirine) 7,8 , alkylation (chlorambucil) 9,10 , in-line probing 11 , and SHAPE 12 , and these have emerged as useful tools to identify RNA-ligand interactions in vitro and recently in cultured cells 13 . Although elegant, application of these methods in vivo is potentially limited by transcriptional effects during prolonged probe treatment 14 , cellular damage by ultraviolet irradiation 15,16 , and nucleobase biases [17][18][19] .…”