Extending the Sensitivity of CEST NMR Spectroscopy to Micro‐to‐Millisecond Dynamics in Nucleic Acids Using High‐Power Radio‐Frequency Fields

Abstract

Biomolecules undergo motions on the micro‐to‐millisecond timescale to adopt low‐populated transient states that play important roles in folding, recognition, and catalysis. NMR techniques, such as Carr–Purcell–Meiboom–Gill (CPMG), chemical exchange saturation transfer (CEST), and R1ρ are the most commonly used methods for characterizing such transitions at atomic resolution under solution conditions. CPMG and CEST are most effective at characterizing motions on the millisecond timescale. While some implementations of the R1ρ experiment are more broadly sensitive to motions on the micro‐to‐millisecond timescale, they entail the use of selective irradiation schemes and inefficient 1D data acquisition methods. Herein, we show that high‐power radio‐frequency fields can be used in CEST experiments to extend the sensitivity to faster motions on the micro‐to‐millisecond timescale. Given the ease of implementing high‐power fields in CEST, this should make it easier to characterize micro‐to‐millisecond dynamics in biomolecules.

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

“…The intensities were normalized to the average intensity of the three zero delay measurements. Exchange parameters were then obtained by fitting experimental intensity values to numerical solutions of the B-M equations and RF field inhomogeneity was taken into account during CEST fitting as described previously 59 . No equilibration of GS magnetization was assumed when integrating the B-M equations for non-methyl probes 59 , while equilibration was assumed for the methyl CEST given that the sequence employs non-selective hard pulses.…”

“…Exchange parameters were then obtained by fitting experimental intensity values to numerical solutions of the B-M equations and RF field inhomogeneity was taken into account during CEST fitting as described previously 59 . No equilibration of GS magnetization was assumed when integrating the B-M equations for non-methyl probes 59 , while equilibration was assumed for the methyl CEST given that the sequence employs non-selective hard pulses. Fits of CEST data were carried out assuming unequal R2 or assuming equal R2 for duplex melting 21 and other ES measurements, respectively.…”

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

“…Treatment of spin-lock inhomogeneity and alignment of the initial magnetization during CEST fitting was performed as described previously 48 . Only GS magnetization was considered to be present at the start of the relaxation delay during CEST fitting for the C2/C6/C8/C1’ and imino nitrogen spins, owing to the absence of the equilibration delay in the pulse sequence.…”

delta-Melt: Nucleic acid conformational penalties from melting experiments

“…7). 13 C/ 15 N CEST 13 C/ 15 N CEST experiments on the aromatic C6/C8/C2 and C1' carbons, and imino nitrogen U-N3 atoms were carried out using a pulse sequence employing a selective excitation scheme in a 1D manner, as described previously 22,47,48 . The 13 C CEST experiments on the methyl carbon in hpGGACU m6A6 (m 6 A6 C10 lb) were carried out in a 2D mode using a pulse sequence derived from prior 13 C CEST pulse sequences 21,47,49 .…”

“…The analysis of CEST data was performed as described previously 22,48 . Briefly, 1D peak intensities were obtained in a manner similar to that for R1ρ.…”