Quantitative Label-Free Surface-Enhanced Raman Spectroscopic Transcriptome-Wide Profiling of RNA Adenosine Methylation

RNA N 6 -methyladenosine (m 6 A) modification plays critical roles in diverse biological processes and human diseases, but its functional studies are greatly impeded by the inability to quantify the m 6 A methylation in whole-transcriptomes. Here, An integrated label-free surface-enhanced Raman spectroscopy (SERS) profiling strategy, named m 6 A-SERS-profiler, is developed for quantitative transcriptome-wide m 6 A detection of cellular and serum RNA. The m 6 A-SERS-profiler is rationally designed through coupling the plasmonic liquid microparticle-based label-free SERS biosensing with the custom deep learning-assisted spectral analysis of m 6 A methylation signatures. With this m 6 A-SERS-profiler, direct identification of SERS spectral signatures of canonical nucleoside monophosphates and N 6 -methyladenosine 5′-monophosphate is achieved. The applicability of the m 6 A-SERS-profiler is verified by quantifying the m 6 A methylation status of microRNA and natural RNA in total RNA isolated from cancer cells and clinical serums. The results reveal a significant difference in the m 6 A RNA methylation among breast cancer cells of different subtypes and between clinical serums from healthy individuals and breast cancer patients. Moreover, the method can quantitatively track the m 6 A dynamics in cancer cells caused by pharmacological inhibition of the m 6 A methyltransferase. Thus, this m 6 A-SERS-profiler holds great promise for transcriptome-wide quantitative detection of m 6 A RNA methylation to investigate biological functions and dynamics of m 6 A methylation in human diseases.