Detection and measurement of RNA modifications through targeted RNA editing
N6-methyladenosine, m6A, is the most frequent post-transcriptional modification in RNA that has been implicated in nearly every aspect of the RNA life cycle. Detecting m6A RNA mN6-methyladenosine, m6A, is the most frequent internal post-transcriptional modification in mRNA, and it has been implicated in nearly every aspect of the RNA life cycle. Furthermore, proteins involved in the formation, removal, and recognition of m6A have emerged as critical regulators of a variety of physiological processes, ranging from stem cell proliferation to learning and memory. Thus, detecting m6A RNA methylation sites in cells is critical for our ability to understand how this modification impacts gene expression to contribute to cellular function and disease states. Currently available m6A site mapping and quantification techniques rely heavily on the immunoprecipitation of methylated RNAs using m6A-recognizing antibodies. However, this antibody-dependent approach has limitations including cross-reactivity of the anti-m6A antibodies, the requirement for large RNA input, time-consuming library preparation, and high cost. The current invention fulfills the need for a simple, sensitive, and highly specific antibody-free method for single-nucleotide resolution mapping and quantification of m6A in the transcriptome.ethylation sites in cells is critical for our ability to understand how this modification impacts gene expression to contribute to cellular function and disease states. Current available m6A site mapping and quantification techniques are heavily reliant on the immunoprecipitation of methylated RNAs using m6A-recognizing antibodies. However, this antibody-dependent approach has various limitations such as the cross-reactivity of the anti-m6A antibodies, requirement for large RNA input for immunoprecipitation and library preparation, time-consuming library preparation step, and high costly nature of the antibody-based assays. Thus, there is a need for a simple, sensitive, and more specific antibody-free method for single-nucleotide resolution mapping and quantitation of m6A in the transcriptome.
Dr. Kathryn Meyer and colleagues have developed a strategy to detect RNA modifications, including m6A, that is amenable to in vivo, cellular and in vitro assays. Their method involves using the APOBEC1 cytidine deaminase domain to edit m6A-adjacent cytidines, which can be detected using RNA-seq, reverse transcription and Sanger Sequencing. This approach substantially improves the time and cost associated with global m6A detection and enables transcriptome-wide mapping of m6A in limited RNA samples, without the need for m6A antibodies. Their strategy can be further extended to detect other RNA modifications by fusing APOBEC1 to alternative protein domains. Therefore, this invention is a powerful new method with has broad utility in the detection of RNA modifications and is suitable for use in in vivo, cellular, and in vitro assays.
- Inexpensive, simple, sensitive, antibody-free method for global m6A detection and quantification
- Can disgtinguish m6A sites from the structurally similar m6Am sites
- Provides single-nucleotide resolution and also single cell resolution
- Broad utility in detecting RNA modifications for the rapidly growing field of epigenomics