N-6-Allyladenosine: A New Small Molecule For Rna Labeling Identified By Mutation Assay
Journal of the American Chemical Society(2017)
摘要
RNA labeling is crucial for the study of RNA structure and metabolism. Herein we report N-6-allyladenosine (a(6)A) as a new small molecule for RNA labeling through both metabolic and enzyme-assisted manners. a(6)A behaves like A and can be metabolically incorporated into newly synthesized RNAs inside mammalian cells. We also show that human RNA N-6-methyladenosine (m(6)A) 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 N-6-position of A within specific sequence to generate a(6)A-labeled RNAs. The iodination of N-6-allyl group of a(6)A under mild buffer conditions spontaneously induces the formation of N-1,N-6-cyclized adenosine and creates mutations at its opposite site during complementary DNA synthesis of reverse transcription. The existing m(6)A in RNA is inert to methyltransferase-assisted allyl labeling, which offers a chance to differentiate m(6)A 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.
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