NMR Chemical Exchange Measurements Reveal That N6-Methyladenosine Slows RNA Annealing

N-6-Methyladenosine (m(6)A) is an abundant epitranscriptomic modification that plays important roles in many aspects of RNA metabolism. While m(6)A is thought to mainly function by recruiting reader proteins to specific RNA sites, the modification can also reshape RNA-protein and RNA-RNA interactions by altering RNA structure mainly by destabilizing base pairing. Little is known about how m(6)A and other epitranscriptomic modifications might affect the kinetic rates of RNA folding and other conformational transitions that are also important for cellular activity. Here, we used NMR R-1 rho relaxation dispersion and chemical exchange saturation transfer to noninvasively and site-specifically measure nucleic acid hybridization kinetics. The methodology was validated on two DNA duplexes and then applied to examine how a single m(6)A alters the hybridization kinetics in two RNA duplexes. The results show that m(6)A minimally impacts the rate constant for duplex dissociation, changing k(off) by similar to 1-fold but significantly slows the rate of duplex annealing, decreasing k(on) by similar to 7-fold. A reduction in the annealing rate was observed robustly for two different sequence contexts at different temperatures, both in the presence and absence of Mg2+. We propose that rotation of the N-6-methyl group from the preferred syn conformation in the unpaired nucleotide to the energetically disfavored anti conformation required for Watson-Crick pairing is responsible for the reduced annealing rate. The results help explain why in mRNA m(6)A slows down tRNA selection and more generally suggest that m(6)A may exert cellular functions by reshaping the kinetics of RNA conformational transitions.