Unmasking DNA Resonances by Suppression of Hyperpolarized Water.

Buffers based on hyperpolarized water (HyperW) generated by dissolution dynamic nuclear polarization (dDNP) enable orders-of-magnitude signal enhancements in biomolecular NMR and residue-resolved access to a range of target systems at near-physiological concentrations and conditions. At the same time, the benefits of this signal enhancement are fundamentally counteracted by an overwhelming water signal that obscures most of the 1H spectrum. Therefore, nonisotopically enriched targets, including most nucleic acids, the second-most-abundant class of biomolecules, remain largely inaccessible to dDNP applications. Here, we introduce a versatile postprocessing strategy based on singular value decomposition that selectively removes the obscuring HyperW contribution while preserving full biomolecular hyperpolarization. This approach eliminates this major downside of biomolecular dDNP experiments in aqueous environments and restores access to the largest share of the 1H spectral range. Our HyperW signal suppression method enabled us to access previously masked hyperpolarization reservoirs across a range of DNA targets. (i) We were able to monitor multiple site-resolved polarization transfers from HyperW to DNA via exchange-relayed NOE pathways in real time, in a single experiment, for all DNA moieties (aromatic, amino, carbohydrate) distributed across the full range of the 1H spectrum. In contrast, hyperpolarized NMR was previously largely limited to imino resonances. (ii) Application to noncanonical, structurally distinct i-motif and G-quadruplex DNAs allowed us to selectively hyperpolarize distinct molecular regions, providing real-time insight into solvent interactions that are invisible to conventional NMR. Finally, the method is broadly applicable, as shown with five diverse target molecules, and thus provides a versatile route to biomolecular 1H-detected dDNP in aqueous environments.