Mapping small molecule–RNA binding sites via Chem-CLIP synergized with capillary electrophoresis and nanopore sequencing

Target validation and identification of binding sites are keys to the development of bioactive small molecules that target RNA. Herein, we describe optimized protocols to profile small molecule-RNA interactions and to define binding sites of the small molecules in RNAs using covalent chemistry. Various reactive modules appended to an RNA-binding small molecule were studied for cross-linking to the RNA target. Electrophilic modules, whether N-chloroethyl aniline or diazirine, have reactive profiles consistent with induced proximity; however, probes with N-chloroethyl aniline were more reactive and more specific than those with a diazirine cross-linking moiety. Depending upon the identity of the cross-linking module, covalent adducts with different nucleotides that are proximal to a small molecule's binding site were formed. The nucleotides where cross-linking occurred were elucidated by using two different platforms: (i) automated capillary electrophoresis that identified a binding site by impeding reverse transcriptase, or "RT stops"; and (ii) nanopore sequencing where the cross-link produces mutations in the corresponding complementary DNA formed by reverse transcriptase-polymerase chain reaction amplification of the cross-linked RNA. These approaches are broadly applicable to aid in the advancement of chemical probes targeting RNA, including identifying binding sites and using covalent chemistry to screen for RNA-binding molecules in a high throughput format.