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.

Center for Molecular Medicine Cologne Faculty of Medicine and University Hospital Cologne University of Cologne 50931 Cologne Germany

Central European Institute of Technology Masaryk University 625 00 Brno Czech Republic

Cologne Excellence Cluster on Cellular Stress Responses in Aging Associated Diseases University of Cologne and University Hospital Cologne 50931 Cologne Germany

Department of Translational Genomics Faculty of Medicine and University Hospital Cologne University of Cologne 50931 Cologne Germany

Institute of Biological Chemistry Faculty of Chemistry University of Vienna Währinger Str 38 1090 Vienna Austria

Institute of Human Genetics University Hospital Cologne 50931 Cologne Germany

National Centre for Biomolecular Research Faculty of Science Masaryk University Kamenice 5 625 00 Brno Czech Republic

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