RNA Modification

Applications involving RNA modification include isolation of RNA species by enrichment or depletion, structural biophysical studies, in situ hybridization analyses, cellular localization, identification of RNA aptamers, RNA mapping, single nucleoside generation for liquid chromatography-mass spectrometry (LC-MS), RNase protection assays, preparing RNA for cloning, and research into the construction of modification patterns in long, complex RNAs for the development of RNA-based therapeutics and vaccines.

The dynamics of RNA modifications and cellular epigtranscriptomes are active areas of research with demonstrated clinical applications. Investigations into the biological functions of modified RNA species and development of RNA-based therapeutics encompass mRNAs, microRNAs, siRNAs, aptamers, and oligonucleotides. A vast number of modifications to RNA species occur in nature, many of which can be manipulated to confer in vitro or in vivo functionalities. RNA modifications can enhance stability, catalytic, and binding properties. Tagged or labeled RNA can be useful in detection and enrichment approaches.

There are many examples in nature of modified RNAs, for instance, in eukaryotic organisms, mRNAs are characteristically modified by the post-transcriptional addition of an inverted 7-methyl-G to form a cap structure that is critical for the maintenance of mRNA stability and for ensuring engagement of the mRNA translation machinery.  Another example of modification occurs on the 3´-ends of some classes of small regulatory RNAs (piwi interacting RNAs and endogenous small interfering RNAs in animals, and plant microRNAs) are modified by the addition of a methyl group to the 2´-position of the ribose on the terminal nucleotide. This modification is essential for piRNA stability and regulatory function.

Enzymes that modify RNA are important tools to characterize RNA species, because their activity can differ based upon existing modifications. For instance, 5´-capped RNAs are not substrates for polynucleotide kinases, or RNA ligases, but are substrates for decapping enzymes. Similarly, ligation and polyadenylation efficiency for 2´-O-methylated 3´-ends of RNAs differs from those with unmodified 3′-ends. The selective properties of RNA modifying enzymes enable researchers to distinguish these RNA species. Some unique end modifications can be used to selectively degrade or isolate particular RNA species when treated with the correct series of enzymes.

Modification in vitro of RNA ends can be important to prepare RNA for downstream applications such as adaptor ligations in preparation for sequencing platforms where certain end modifications are required for efficient representation.

Modification of RNA bases, e.g., 5 methyl cytosine, or N6 methyladenine, to evade innate immune responses in mRNA transfected cells is important in experiments that use this technique to influence cell fate.

RNA modifications can be identified and quantified using single nucleosides optimally generated with the Nucleoside Digestion Mix (NEB #M0649S) for liquid chromatography–mass spectrometry (LC-MS) analysis. Alternatively, site-specific nucleases can be used to generate fragments for analysis by LC-MS [1,2].

References:
1.    Wolf et al.(2022) Nucleic Acids Research, 50,18, e106.  
2.    Wolf et al.(2023) ACS Pharmacology & Translational Science, 6, 618-630.

RNA modification analysis by liquid chromatography-mass spectrometry (LC-MS)