RNA Crosslinking and Click Chemistry with Platinum Compounds

Methods to assess three-dimensional RNA structure inside and outside of the cell are of great importance. Crosslinking reagents that report on tertiary interactions in RNA are of particular interest as constraints for structure models and as reporters for RNA dynamics. Small, square-planar Pt(II) compounds such as cisplatin are well-known anticancer therapeutics that bind purine nucleobases. While well-studied in DNA, the interaction properties of these compounds with complex RNA structures are less understood. Here we present studies exploring Pt(II) crosslinking in structured RNAs, including a ribozyme and a ribosomal subdomain. In order to isolate and label Pt-bound RNAs, we have developed azide-modified Pt(II) compounds that undergo ‘click’ chemistry with functionalized fluorophores, biotin, and other reagents. Mapping sites following in vivo exposure demonstrates that Pt(II) preferentially targets purine-rich loops, and fluorescence labeling demonstrates broad binding of these reagents to different cellular RNAs. Taken together, Pt(II) reagents present new opportunities for RNA structure analysis both in vitro and in vivo.