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

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.