meCLICK-Seq – a substrate-hijacking and RNA degradation strategy for the study of RNA methylation

The fates of RNA species in a cell are controlled by ribonucleases, which degrade them by exploiting the universal structural 2′-OH group. This phenomenon plays a key role in numerous transformative technologies, for example, RNA interference and CRISPR/Cas13-based RNA editing systems. These approaches, however, are genetic or oligomer-based and so have inherent limitations. This has led to interest in the development of small molecules capable of degrading nucleic acids in a targeted manner.

University of Cambridge researchers now describe click-degraders, small molecules that can be covalently attached to RNA species through click-chemistry and can degrade them, that are akin to ribonucleases. By using these molecules, the researchers have developed the meCLICK-Seq (methylation CLICK-degradation Sequencing) a method to identify RNA modification substrates with high resolution at intronic and intergenic regions. The method hijacks RNA methyltransferase activity to introduce an alkyne, instead of a methyl, moiety on RNA. Subsequent copper(I)-catalyzed azide–alkyne cycloaddition reaction with the click-degrader leads to RNA cleavage and degradation exploiting a mechanism used by endogenous ribonucleases. Focusing on N⁶-methyladenosine (m⁶A), meCLICK-Seq identifies methylated transcripts, determines RNA methylase specificity, and reliably maps modification sites in intronic and intergenic regions.

Proposed mechanism of action of meCLICK-Seq

The researchers show that METTL16 deposits m⁶A to intronic polyadenylation (IPA) sites, which suggests a potential role for METTL16 in IPA and, in turn, splicing. Unlike other methods, the readout of meCLICK-Seq is depletion, not enrichment, of modified RNA species, which allows a comprehensive and dynamic study of RNA modifications throughout the transcriptome, including regions of low abundance. The click-degraders are highly modular and so may be exploited to study any RNA modification and design new technologies that rely on RNA degradation.