NMR Chemical Exchange Measurements Reveal That <i>N</i>⁶-Methyladenosine Slows RNA Annealing

Abstract

<i>N</i>⁶-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 <i>R</i>_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 <i>k</i>_off by ∼1-fold but significantly slows the rate of duplex annealing, decreasing <i>k</i>_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 <i>N</i>⁶-methyl group from the preferred <i>syn</i> conformation in the unpaired nucleotide to the energetically disfavored <i>anti</i> 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.