Heavy Water Remodels the DNA Energy Landscape to Stabilize Folded States and Slow Transitions

Heavy water (D₂O) subtly strengthens hydrogen bonds, yet its full biological impact has remained fragmentary and controversial. Here we present an integrated, multi-scale study showing that D₂O consistently fortifies DNA while dampening life processes. From bulk Escherichia coli (E. coli) cultures to PCR assays, circular dichroism melting, FRET measurements, and single-molecule force spectroscopy, every method converges on the same picture: replacing H₂O with D₂O stabilizes both canonical duplexes and noncanonical i-motif structures, elevates thermal and mechanical thresholds for strand separation, and translates into markedly slower bacterial growth. Notably, our first direct measurements of hairpin unzipping and refolding forces in heavy water show that deuterium-enhanced hydrogen bondingrather than D₂O's higher viscositydominates the slowdown of DNA-related transitions, deepening the folded-state well while only modestly stabilizing intermediates. A minimal quantum-mechanical analysis attributes this trend to reduced zero-point energies of hydrogen-bond vibrations upon H → D substitution. This coherence across independent techniques clarifies longstanding inconsistencies surrounding heavy-water effects and nucleic-acid mechanics. By showing how isotopic substitution reshapes the DNA energy landscape, our work provides a quantitative foundation for future studies that exploit deuterium to probe or control biomolecular function.