Folding and unfolding dynamics of a DNA aptamer studied by heteronuclear ¹H-¹³C correlation zz-exchange spectroscopy

Single-stranded DNA (ssDNA) molecules can fold into compact functional structures, yet their conformational dynamics remain poorly understood at atomic resolution. Here, we use heteronuclear ¹H-¹³C correlation zz-exchange NMR spectroscopy to investigate the folding and unfolding kinetics of the 26-nucleotide DNA aptamer NU172, which recognizes and inhibits thrombin, an extracellular protein that plays a crucial role in coagulation. NU172 adopts a compact structure involving a G-quadruplex, a reverse Hoogsteen base pair, and Watson-Crick base pairs. At physiological temperature, ¹H-¹³C HSQC spectra revealed signals from both folded and unfolded states in slow exchange on the NMR chemical-shift timescale, whereas ¹H-¹⁵N HSQC spectra detected only the folded state because the unfolded state lacks protected imino protons. Through non-TROSY and aromatic ¹³C TROSY versions of heteronuclear ¹H-¹³C zz-exchange experiments implemented for uniformly ¹³C,¹⁵N-labeled nucleic acids, we observed exchange cross peaks between the folded and unfolded states and quantitatively characterized the folding/unfolding dynamics of NU172. The exchange rate was ∼0.6 s⁻¹ at 35 °C, with the unfolded state populated at ∼30%. This study demonstrates that heteronuclear ¹H-¹³C correlation zz-exchange spectroscopy is well suited for atomic-level characterization of functional folded ssDNA molecules such as aptamers and DNAzymes.