There are tens of thousands of different RNA molecules in the cell, most of which have unknown structure and function. A growing number of them have been directly connected with human disease. These RNAs interact with each other and with proteins and small molecules, and control gene expression and cellular pathways in complex ways. Assessing locations, sequences, structures, and functions of these RNAs will become increasingly important to medicine as well as basic biology. Inspired by these facts, we are developing molecular tools to aid the study of cellular RNAs. As a strategy for mapping RNA structures in vivo, we have developed cell-permeable reagents that contain reactive acylimidazole groups. These enter the cell and acylate 2’-hydroxyl groups at positions where they are accessible to solvent. The acylation allows mapping of structure by reverse transcriptase-mediated primer extension. Combining this with RNA sequencing methodologies has allowed us with collaborators to map the structures of large numbers of RNAs in mammalian cells, and this work is uncovering interesting data about protein-RNA interactions and RNA modifications that occur in living systems. The selective acylation of RNA 2’-OH groups is further being developed as a strategy for controlling RNA function. In vitro reaction of soluble azide-functionalized acylimidazoles with RNAs can result in high levels of acylation at over 50% of the 2’-OH groups in an RNA strand. This polyacylation (termed ”cloaking”) strongly inhibits the ability of the RNA to hybridize and fold. At a selected time, the RNA can be de-acylated (”uncloaked”) rapidly and in high yield by addition of water-soluble phosphines, triggering RNA folding and hybridization. This represents a new way to control and switch RNA structure and function. Signficantly, it can be applied postsynthetically to short and long RNAs prepared either chemically or enzymatically. We expect that this strategy can be useful as a technology for timed and controlled activation of RNAs in vitro, and ultimately as a method for switching on RNAs in living cells for study of their downstream biological effects.

Small-molecule covalent probes for study and control of RNA

255th National Meeting (2018)

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