Sequencing-free Analysis of Multiple Methylations on Gene-Specific mRNAs

Research output: Journal Publications and Reviews (RGC: 21, 22, 62)21_Publication in refereed journalpeer-review

2 Scopus Citations
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Original languageEnglish
Pages (from-to)6010–6018
Journal / PublicationJournal of the American Chemical Society
Issue number13
Online published23 Mar 2022
Publication statusPublished - 6 Apr 2022


RNA epigenetics is a new layer of mechanism to regulate gene expression, but limited techniques are available to profile the status of mRNA modifications. Here, we describe a molecule proximity-based technique for simultaneous analysis of multiple types of mRNA methylation with specific gene information in living cells. N6-methyladenosine (m6A) or N1-methyladenosine (m1A) modifications on multiple mRNAs can be individually or simultaneously analyzed. A chip fabricated with vertically aligned, high-aspect-ratio diamond nanoneedles was used to access the intracellular domain in a minimum-invasive format and to isolate the mRNAs out of the cell cytoplasm while keeping cells alive. In the subsequent on-chip analytical procedures, the isolated RNAs were encoded, amplified, and visualized to derive a quantitative measurement of the associated gene-specific m6A or m1A modifications. Notably, a proximity ligation approach was developed to resolve dual methylation on an individual mRNA segment. Using this method, we investigated the dynamics of mRNA methylation in mammalian cells under physical or chemical stimuli and showed that m6A and m1A in mRNAs are heavily involved in the cellular stress response. Our results also suggested the common existence of single m6A modification in the basigin (BSG) mRNA but a rare occurrence of m6A and m1A dual methylation in the same BSG transcript.