RNA is a versatile biomolecule with a broad range of biological functions that go far beyond its initially described role as a simple information carrier. The development of chemical methods to control, manipulate and modify RNA has the potential to yield new insights into its many functions and properties. Traditionally, most of these methods involved the chemical modification of RNA structure using solid-state synthesis or enzymatic transformations. However, over the past 15 years, the direct functionalization of RNA by selective acylation of the 2′-hydroxyl (2′-OH) group has emerged as a powerful alternative that enables the simple modification of both synthetic and transcribed RNAs. In this Review, we discuss the chemical properties and design of effective reagents for RNA 2′-OH acylation, highlighting the unique problem of 2′-OH reactivity in the presence of water. We elaborate on how RNA 2′-OH acylation is being exploited to develop selective chemical probes that enable interrogation of RNA structure and function, and describe new developments and applications in the field.
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The authors thank the U.S. National Institutes of Health (GM127295 and GM130704) for grant support.
The authors declare no competing interests.
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- Non-coding RNAs
(ncRNAs). RNA molecules that are not translated into proteins, but often have other biological roles, such as assisting in splicing, gene regulation and DNA replication.
- Transfer RNA
(tRNA). A non-coding RNA molecule that carries an amino acid and helps to decode messenger RNA (mRNA) into protein; tRNAs contain a three-nucleotide sequence (anticodon) that matches to a three-nucleotide sequence on mRNA (codon).
- Messenger RNAs
(mRNAs). Coding RNA molecules that convey the genetic information from DNA to facilitate biosynthesis of functional proteins; the mRNA nucleotide sequence is translated into protein by the ribosome.
- Psoralen analysis of RNA interactions
(PARIS). A method for mapping RNA structure in cells using the small-molecule psoralen as an RNA crosslinker; crosslinked RNA fragments are analysed with next-generation sequencing and, using informatics methods, duplex regions can be assigned throughout the transcriptome.
- Crosslinking immunoprecipitation
(CLIP). A method for studying protein–RNA interactions whereby cells are exposed to high-intensity ultraviolet light, which crosslinks proteins and RNA molecules that are in close proximity; using immunoprecipitation, the complexes can be isolated and RNAs can be identified with sequencing.
- Selective 2′-hydroxyl acylation analysed by primer extension
(SHAPE). A method for analysing RNA structure whereby 2′-OH groups in RNA can be acylated with small-molecule reagents in unpaired, accessible and flexible regions; the acylation groups block reverse transcriptase during primer extension. Subsequent analysis of primer extension products is used to predict accessible regions and secondary structures of RNAs.
- Dimethyl sulfate footprinting
(DMS footpinting). A method for determining unpaired regions of nucleic acids using DMS, which can methylate the N1 position of adenine and N3 position of cytosine; methylation occurs selectively on unpaired nucleobases and can block reverse transcriptase. Analysis of reverse-transcription products reveals unpaired regions in nucleic acids.
- Reverse transcriptases
A class of DNA polymerase enzymes that produces complementary DNA (cDNA) from an RNA template.
- Next-generation sequencing
(NGS). A term used to describe different modern sequencing technologies, all of which are capable of determining the sequence of millions of DNA fragments in a single reaction volume.
An RNA molecule that can carry out an enzymatic function, such as ligation or hydrolysis reactions.
- Viral RNA
RNA that defines the genetic material of a virus; this can be single-stranded or double-stranded in structure.
- Prebiotic RNA synthesis
Refers to part of the ‘RNA World’ hypothesis that suggests that RNA molecules proliferated before DNA and proteins and relied on self-replication.
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Velema, W.A., Kool, E.T. The chemistry and applications of RNA 2′-OH acylation. Nat Rev Chem 4, 22–37 (2020). https://doi.org/10.1038/s41570-019-0147-6
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