RNA Cloaking by Reversible Acylation

Abstract

We describe a selective and mild chemical approach to controlling RNA hybridization, folding, and enzyme interactions. Reaction of RNAs in aqueous buffer with an azide-substituted acylating agent (100-200 mM) yields several 2′-OH acylations per RNA strand in as little as 10 min. This poly-acylated (“cloaked”) RNA is strongly blocked from hybridization with complementary nucleic acids, from cleavage by RNA-processing enzymes, and from folding into active aptamer structures. Importantly, treatment with a water-soluble phosphine triggers a Staudinger reduction of the azide groups, resulting in spontaneous loss of acyl groups (“uncloaking”). This fully restores RNA folding and biochemical activity.

Conditional control of RNA-guided nucleic acid cleavage and gene editing

“…Traditionally, the conjugation of chemical moiety to the oligonucleotides may interfere the access of other biomolecules 38 . Given ubiquitous 2′-OH groups in RNA ribose, the protection of such groups will constitute an initial step 34 . The gRNA function was expected to be temporarily suspended by attaching the AMN groups, which can be removed by treatment with phosphine through a Staudinger reduction 24,34 .…”

“…Given ubiquitous 2′-OH groups in RNA ribose, the protection of such groups will constitute an initial step 34 . The gRNA function was expected to be temporarily suspended by attaching the AMN groups, which can be removed by treatment with phosphine through a Staudinger reduction 24,34 . Hence, we were interested in exploring how the post-synthetic masking and chemically selective unmasking of gRNA can be applied to reprogram CRISPR systems.…”

“…2) 30 . An initial study was then performed to chemically mask this gRNA construct 34 . After quenching the reaction, the gRNA products were recovered by ethanol precipitation and subjected to denaturing electrophoresis.…”

“…Recently, RNA engineering for chemical and biological applications has emerged as an exciting research field 31–33 . In a recent study, Kool and colleagues have developed a chemical strategy that achieved robust blockage of RNA structure by chemical acylation, and efficient recovery of RNA structure with a subsequent de-acylation procedure 34 . This strategy is promising and we envisioned that it will benefit the development of chemical switches to controlling CRISPR systems.…”

Photocontrol of CRISPR/Cas9 function by site-specific chemical modification of guide RNA

“…Our design of site-specic photoactivable gRNA presented a new pathway for modication of RNA based on the RNA-protein interaction rather than RNA itself. More than optical control, these specic sites could be also modied with other stimuliresponsive groups, 54,56,57 which would potentially further enrich the toolbox for conditional control of CRISPR-Cas9 function in the future.…”