Magnetization Transfer to Enhance NOE Cross‐Peaks among Labile Protons: Applications to Imino–Imino Sequential Walks in SARS‐CoV‐2‐Derived RNAs

Abstract

2D NOESY plays a central role in structural NMR spectroscopy. We have recently discussed methods that rely on solvent‐driven exchanges to enhance NOE correlations between exchangeable and non‐exchangeable protons in nucleic acids. Such methods, however, fail when trying to establish connectivities within pools of labile protons. This study introduces an alternative that also enhances NOEs between such labile sites, based on encoding a priori selected peaks by selective saturations. The resulting selective magnetization transfer (SMT) experiment proves particularly useful for enhancing the imino–imino cross‐peaks in RNAs, which is a first step in the NMR resolution of these structures. The origins of these enhancements are discussed, and their potential is demonstrated on RNA fragments derived from the genome of SARS‐CoV‐2, recorded with better sensitivity and an order of magnitude faster than conventional 2D counterparts.

The Extended Hadamard Transform: Sensitivity‐Enhanced NMR Experiments Among Labile and Non‐Labile 1Hs of SARS‐CoV‐2‐derived RNAs

“…[26] As 1 H assignments for this RNA fragment have been reported, [27] experiments were carried out mainly to examine the presence of artifacts and relative sensitivities. As already demonstrated in Ref [14] the most basic of these experiments, SMT, will significantly enhance cross-relaxation peaks when compared to its conventional counterpart; under the assayed conditions (grey vs black traces in Figure 4), ca. 4-and 6-fold SNR enhancements per unit time (SNRT) were observed for iminoimino and imino-amino proton correlations, respectively.…”

“…This was illustrated in ref. [14], which showed that although HMT could provide reliable cross-peaks between different spin pools, it will in general fail when connectivities are sought within the same pool of spins -e. g., when seeking imino-imino correlations within nucleic acids. This problem originates from Hadamard assumption of a linear independence between the weightings imparted to the various peaks during its frequency encoding.…”

“…We have recently demonstrated a number of avenues whereby solvent exchanges can be used to enhance correlations involving labile protons. [ 11 , 12 , 13 , 14 ] For instance, instead of a single t 1 encoding followed by a mixing time, the peaks of interest can be encoded and cross‐correlated by looping multiple encoding/mixing times. [11] During each mixing time, labile protons are then repolarized through exchange processes with an unperturbed water 1 H magnetization, acting as a source for a more efficient polarization transfer.…”

“…If carried out with continuous irradiations this may be considered as a special case of the selective looped manipulations, as labile repolarizations by solvent exchanges will occur continuously rather than discretely during the inter‐loop delays, leading for instance to selective magnetization transfer (SMT) experiments. [14] Although lacking Fourier multiplexing these experiments can, in analogy to Saturation Transfer Experiments (STD), [15] deliver NOE cross‐peaks showing an enhanced sensitivity compared to conventional methods. Further enhancements of such correlations can be achieved by incorporating into SMT a Hadamard scheme,[ 16 , 17 , 18 ] whereby monochromatic frequency‐selective pulses are replaced by polychromatic saturation or polychromatic inversion pulse blocks, perturbing multiple labile peaks in what will become the F 1 frequency domain.…”

Conformational Ensembles of Noncoding Elements in the SARS-CoV-2 Genome from Molecular Dynamics Simulations

“… 16 We then proceed by describing our predictions on four elements of increasing complexity ( Figure 1 ) and discuss the agreement with available NMR measurements. 32 , 33 Stem loop 5a (SL5a) is part of the four-way junction SL5 containing the AUG starting codon, whose function is linked to RNA packaging. 34 Stem loop 1 (SL1) has been suggested to play a role in escaping the viral mechanism inhibiting mRNA translation.…”