a⁶A-seq: N ⁶-allyladenosine-based cellular messenger RNA metabolic labelling and sequencing

The integration of RNA metabolic labelling by nucleoside analogues with high-throughput RNA sequencing has been harnessed to study RNA dynamics. The immunoprecipitation purification or chemical pulldown technique is generally required to enrich the analogue-labelled RNAs. Here we developed an a⁶A-seq method, which takes advantage of Nallyladenosine (a⁶A) metabolic labelling on cellular mRNAs and profiles them in an immunoprecipitation-free and mutation-based manner. a⁶A plays a role as a chemical sequencing tag in that the iodination of a⁶A in mRNAs results in 1,N ⁶-cyclized adenosine (cyc-A), which induces base misincorporation during RNA reverse transcription, thus making a⁶A-labelled mRNAs detectable by sequencing. A nucleic acid melting assay was utilized to investigate why cyc-A prefers to be paired with guanine. a⁶A-seq was utilized to study cellular gene expression changes under a methionine-free stress condition. Compared with regular RNA-seq, a⁶A-seq could more sensitively detect the change of mRNA production over a time scale. The experiment of a⁶A-containing mRNA immunoprecipitation followed by qPCR successfully validated the high-throughput a⁶A-seq data. Together, our results show a⁶A-seq is an effective tool to study RNA dynamics.