Sequencing-free Analysis of Multiple Methylations on Gene-SpecificmRNAs br

RNA epigenetics is a new layer of mechanism toregulate gene expression, but limited techniques are available toprofile the status of mRNA modifications. Here, we describe amolecule proximity-based technique for simultaneous analysis ofmultiple types of mRNA methylation with specific gene informationin living cells. N6-methyladenosine (m6A) or N1-methyladenosine(m1A) modifications on multiple mRNAs can be individually orsimultaneously analyzed. A chip fabricated with vertically aligned,high-aspect-ratio diamond nanoneedles was used to access theintracellular domain in a minimum-invasive format and to isolate themRNAs out of the cell cytoplasm while keeping cells alive. In thesubsequent on-chip analytical procedures, the isolated RNAs wereencoded, amplified, and visualized to derive a quantitative measure-ment of the associated gene-specificm6Aorm1A modifications. Notably, a proximity ligation approach was developed to resolve dualmethylation on an individual mRNA segment. Using this method, we investigated the dynamics of mRNA methylation inmammalian cells under physical or chemical stimuli and showed that m6A and m1A in mRNAs are heavily involved in the cellularstress response. Our results also suggested the common existence of single m6A modification in the basigin (BSG) mRNA but a rareoccurrence of m6A and m1A dual methylation in the same BSG transcript.