Hyperpolarized NMR Reveals Low-Populated Folding Intermediates in DNA.

Nuclear magnetic resonance (NMR) spectroscopy is the only biophysical technique capable of characterizing nucleic acid structures at atomic resolution under near-physiological liquid-state conditions. Still, it is fundamentally limited by intrinsically low sensitivity, particularly when analyzing high-molecular-weight, low-abundance, or polymorphic targets, such as DNAs (DNA). In this study, we demonstrate that hyperpolarized aqueous buffers generated via dissolution dynamic nuclear polarization (dDNP) significantly enhance the 1H NMR signals of multiple DNA motifs. The resonances of labile imino and amino protons of DNAs dissolved in hyperpolarized buffers are enhanced up to ∼200-fold and ∼370-fold, respectively. These intense signals serve a 2-fold purpose: (i) as structural fingerprints of DNA folding topologies and (ii) they enable the direct observation of low-populated folding intermediates in DNA polymorphs, such as G-quadruplexes (G4) and i-motifs (iM), which remain undetectable by standard methods. Thus, our findings establish hyperpolarized NMR as a high-sensitivity method for probing DNA structures and folding intermediates across a wide range of motifs, opening possible avenues in liquid biopsy applications and cell-free DNA.