A quantitative model predicts how m⁶A reshapes the kinetic landscape of nucleic acid hybridization and conformational transitions

Abstract

N6-methyladenosine (m6A) is a post-transcriptional modification that controls gene expression by recruiting proteins to RNA sites. The modification also slows biochemical processes through mechanisms that are not understood. Using NMR relaxation dispersion, we show that m6A pairs with uridine with the methylamino group in the anti conformation to form a Watson-Crick base pair that transiently exchanges on the millisecond timescale with a singly hydrogen-bonded low-populated (1%) mismatch-like conformation in which the methylamino group is syn. This ability to rapidly interchange between Watson-Crick or mismatch-like forms, combined with different syn:anti isomer preferences when paired (~1:100) versus unpaired (~10:1), explains how m6A robustly slows duplex annealing without affecting melting via two pathways in which isomerization occurs before or after duplex annealing. Our model quantitatively predicts how m6A reshapes the kinetic landscape of nucleic acid hybridization and conformational transitions, and provides an explanation for why the modification robustly slows diverse cellular processes.

Measuring thermodynamic preferences to form non-native conformations in nucleic acids using melting experiments reveals a rich sequence-specific DNA conformational landscape

“…S4). Assignments for A 6 -RNA m6A16 will be published in a subsequent study (74). These studies also show that the constructs fold into their expected secondary structures as given in Fig.…”

“…for N 6 -methylamino isomerization in m 6 A measured using NMR RD (Fig. 7c) (74). The correlation could be fit with an RMSE of 0.1 kcal/mol and c ሺ i ሻ ~ 0.7 kcal/mol (Fig.…”

“…When paired with uridine, the methylamino group in m 6 A isomerizes between anti (major) and syn (minor) conformations resulting in the loss of a hydrogen-bond (Fig. 7a) (74). This conformational change has been proposed to play roles slowing a variety of biochemical processes that involve duplex melting and annealing (67).…”

“…The site-specifically labeled DNA strands comprising duplexes (A 6 -DNA(A16lb), scaf2_CGT GC (G6lb) and scaf2_TGC GC (G6lb)) were purchased from Yale Keck Oligonucleotide Synthesis Facility with cartridge purification. C2,C8 lb) and hpGGACUm 6 A6(m 6 A6 C10 lb) were synthesized in-house with a MerMade 6 Oligo Synthesizer as described above for the rA16 modified single-strand in A 6 -DNA rA16 , using standard RNA (n-ac-rA, n-ac-rG, n-ac-rC, rU, Chemgenes) and DNA (n-ibu-G, n-bz-A, n-ac-C, T, Chemgenes) phosphoramidites, A phosphoramidites (74).…”

“…Exchange parameters of interest, such as the population of the i th minor conformation (p i ), the exchange rate between the major and i th minor conformation (k ex = k 1 + k -1 , in which k 1 and k -1 are the forward and backward rate constants, respectively), the chemical shift difference between the i th minor and the major conformations (Δω i-Major = Major are the chemical shifts of the nucleus in the i th minor and the major conformations, respectively) were then extracted by fitting the R 1ρ data for a given nucleus to a two-state exchange model using the Bloch-McConnell equations. The validity of a two-state model to analyze the R 1ρ data for Hoogsteen bp formation and N 6 -methyl amino rotation has been extensively validated based on prior studies (7,8,62,63,68,74,81). Owing to the presence of an equilibration delay (5 ms) in the pulse sequences (81,104), initial magnetization at the start of the Bloch-McConnell simulations was assumed to be equilibrated between the major and minor conformations.…”