M6A-SAC-SEQ: A Method For Assaying M6A Epigenetic Modifications Using NGS

SUMMARY

• Epigenetic modifications such as N1-methyladenosine (m1A) can be readily mapped in high throughput using next generation sequencing. Problematically, N6-methyladenosine (m6A) cannot be mapped with these methods. The m6A chemical modification occurs opposite to the Watson-Crick base, which results in a loss of modification information after reverse transcription.
• The inventors developed a chemical method to convert m6A into a homologue of m1A, N6-ethanoadenine, in a targeted and specific fashion. Thus, after chemical modification, m6A sites can then be profiled using reverse transcription and NGS in the same way as m1A.
• The invention is a chemical method of modifying m6A so that this modification can be mapped in high throughput with single base pair resolution using NGS methods. Other epigenetic modifications can already be assayed with NGS, but m6A was not previously compatible with this methodology.
• In proof of concept experiments, m6A sites in the Hela and HEK transcriptomes were mapped at a single base pair resolution and were validated using a lower throughput methodology. Results showed the expected m6A distribution and were validated using a lower throughput m6A profiling method. 

FIGURE

Figure 1 Schematic overview of the M6A-SAC-SEQ methodology showing (1) conversion of m6A to allylic m6A with the MjDim1 enzyme and the allylic donating cofactor, (2) conversion of the allylic m6A to the m1A chemical analogue using I2, (3) using reverse transcription and next generation sequencing to identify locations of the original m6A modifications.

ADVANTAGES

ADVANTAGES

• Adapts current RT-NGS protocols to more epigenetic modifications
• Quantitative base resolution
• Reads out exact modification fractions
• Highly specific to targeted epigenetic modification (10-fold selectivity over background)

APPLICATIONS

• Next generation epigenetic sequencing of m6A
• Whole genome epigenetic profiling

PUBLICATIONS

• Ge R, Ye C, Peng Y, Dai Q, Zhao Y, Liu S, Wang P, Hu L, He C. m6A-SAC-seq for quantitative whole transcriptome m6A profiling. Nat Protoc. 2023 Feb;18(2):626-657. doi: 10.1038/s41596-022-00765-9. Epub 2022 Nov 25. Erratum in: Nat Protoc. 2023 Nov;18(11):3652. PMID: 36434097; PMCID: PMC9918705.

November 1, 2019

Proof of concept

Patent Pending

Licensing,Co-development

Chuan He

• In vivo validation in two cell lines (Hela, HEK)