a6A-seq – N6-allyladenosine-based cellular messenger RNA metabolic labelling and sequencing

In a study published in the journal Fundamental Research, a team of researchers has pioneered a novel method. It incorporated an adenosine analog, N⁶-allyladenosine (a⁶A) into newly transcribed RNAs by nucleoside salvage pathways.

Following the metabolic labeling, the researchers employed a chemical iodination treatment to convert a⁶A within the transcripts into a distinct variant structure. This modified structure can then be accurately identified as base mutation sites using high-throughput sequencing technology.

“We believe that an increasing number of nucleoside analogs will be explored for uses as chemical sequencing tags with the hope of achieving specific labeling on different kinds of RNA, especially mRNA,” says Jianzhao Liu, corresponding author of the study, and a professor at the Institute of Biological Macromolecules at Department of Polymer Science and Engineering of Zhejiang University.

A schematic illustration to study the changes of gene expression profiles
under methionine-free condition by a⁶A-seq

The mutation reads can be used to identify the produced a⁶A-containing transcripts over a time scale. The combination of a⁶A-specific antibody immunoprecipitation and qPCR assay can be applied to validate the high-throughput a⁶A-seq data.

The team found that a⁶A can be recognized by RNA polymerase and incorporated into the newly synthesized cellular mRNA. Meanwhile, the allyl group on a⁶A is bio-orthogonally treated with iodine and a⁶A is converted into a structure of 1,N⁶-cyclized adenosine. Consequently, this modification induces base mismatch during RNA reverse transcription, leading to the generation of A- to C/T mutation reads in subsequent RNA-seq analysis.

At present, an antibody suitable for the immunoprecipitation of a⁶A-containing RNA is commercially available. Leveraging these resources, the researchers used a⁶A in their investigation of alterations in cellular gene expression profiles under methionine-free conditions. Furthermore, they utilized a⁶A-IP as a validation tool to confirm the findings obtained through sequencing. This integrated approach allowed for comprehensive and reliable analysis of gene expression dynamics.

“Our technique will enable researchers to track the newly formed a⁶A-containing transcripts and investigate the dynamics of these transcripts, including RNA synthesis and decay,” says Liu. “a⁶A provides not only a chemical tag for sequencing, but also a tag for antibody enrichment.”

Source – KeAi Communications