eTAM-seq – evolved TadA-assisted N6-methyladenosine sequencing

N⁶-methyladenosine (m⁶A), the most abundant internal messenger RNA modification in higher eukaryotes, serves myriad roles in regulating cellular processes. Functional dissection of m⁶A is, however, hampered in part by the lack of high-resolution and quantitative detection methods. Researchers at the University of Chicago have developed evolved TadA-assisted N⁶-methyladenosine sequencing (eTAM-seq), an enzyme-assisted sequencing technology that detects and quantifies m⁶A by global adenosine deamination. With eTAM-seq, they analyze the transcriptome-wide distribution of m⁶A in HeLa and mouse embryonic stem cells. The enzymatic deamination route employed by eTAM-seq preserves RNA integrity, facilitating m⁶A detection from limited input samples. In addition to transcriptome-wide m⁶A profiling, the researchers demonstrate site-specific, deep-sequencing-free m⁶A quantification with as few as ten cells, an input demand orders of magnitude lower than existing quantitative profiling methods. They envision that eTAM-seq will enable researchers to not only survey the m⁶A landscape at unprecedented resolution, but also detect m⁶A at user-specified loci with a simple workflow.

Global A deamination by TadA8.20

a, Proposed m⁶A detection scheme. TadA8.20 selectively converts A into I, without acting on m⁶A. I is recognized as G by reverse transcriptases. Persistent A post-TadA8.20 treatment corresponds to m⁶A. b,c, In vitro deamination of RNA probes hosting A or m⁶A in ‘CGAUC’ (b) and ‘GGACU’ (c) motifs by TadA8.20. Unmethylated and methylated RNA sequences were prepared through in vitro transcription using ATP and N⁶-methyl-ATP as starting materials, respectively. Treated RNA was reverse transcribed, amplified and subjected to Sanger sequencing. d,e, TadA8.20-catalyzed A-to-I conversion rates in ‘CGAUC’ (d) and ‘GGACU’ (e) probes quantified by next-generation sequencing (NGS). f, Deamination of synthetic A/m⁶A RNA probes by TadA8.20. The 53-nt RNA probes hosting NNANN and NNm⁶ANN motifs were treated by TadA8.20. Deaminated RNA underwent RT and NGS. g, Correlation of persistent A signals captured by eTAM-seq and m⁶A contents in RNA probes. h, Capillary gel electrophoresis analysis of fragmented HeLa mRNA treated with or without TadA8.20 at different temperatures for 3 h. RNA size distribution is plotted on the right. For eTAM-seq, RNA is incubated with TadA8.20 at 53 °C for 1 h followed by a 2-h treatment at 44 °C. Experiments were repeated independently with similar results. i, Transcriptome-wide A-to-I conversion rates in two independent replicates (rep.). Of A sites with ≥100 counts, 10% were randomly sampled to make the scatter plot. Pearson’s r was calculated for all A sites with ≥100 counts. j, Two m⁶A sites in human rRNA. Positions 1829–1835 of 18S rRNA and positions 4217–4223 of 28S rRNA are plotted