N6-Allyladenosine: A New Small Molecule for RNA Labeling Identified by Mutation Assay

J Am Chem Soc. 2017 Dec 6;139(48):17213-17216. doi: 10.1021/jacs.7b06837. Epub 2017 Nov 15.

Abstract

RNA labeling is crucial for the study of RNA structure and metabolism. Herein we report N6-allyladenosine (a6A) as a new small molecule for RNA labeling through both metabolic and enzyme-assisted manners. a6A behaves like A and can be metabolically incorporated into newly synthesized RNAs inside mammalian cells. We also show that human RNA N6-methyladenosine (m6A) methyltransferases METTL3/METTL14 can work with a synthetic cofactor, namely allyl-SAM (S-adenosyl methionine with methyl replaced by allyl) in order to site-specifically install an allyl group to the N6-position of A within specific sequence to generate a6A-labeled RNAs. The iodination of N6-allyl group of a6A under mild buffer conditions spontaneously induces the formation of N1,N6-cyclized adenosine and creates mutations at its opposite site during complementary DNA synthesis of reverse transcription. The existing m6A in RNA is inert to methyltransferase-assisted allyl labeling, which offers a chance to differentiate m6A from A at individual RNA sites. Our work demonstrates a new method for RNA labeling, which could find applications in developing sequencing methods for nascent RNAs and RNA modifications.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine / analogs & derivatives*
  • Adenosine / metabolism
  • Biological Assay*
  • Humans
  • Methyltransferases / metabolism
  • Mutation*
  • RNA / genetics*
  • RNA / metabolism*
  • S-Adenosylmethionine / metabolism
  • Staining and Labeling / methods

Substances

  • N-allyladenosine
  • RNA
  • S-Adenosylmethionine
  • METTL14 protein, human
  • Methyltransferases
  • METTL3 protein, human
  • Adenosine