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

N⁶-Methyladenosine (m⁶A) is an abundant epitranscriptomic modification that plays important roles in many aspects of RNA metabolism. While m⁶A 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 m⁶A 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_1ρ 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 m⁶A alters the hybridization kinetics in two RNA duplexes. The results show that m⁶A minimally impacts the rate constant for duplex dissociation, changing k_off by ∼1-fold but significantly slows the rate of duplex annealing, decreasing k_on by ∼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 N⁶-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 m⁶A slows down tRNA selection and more generally suggest that m⁶A may exert cellular functions by reshaping the kinetics of RNA conformational transitions.