1H R1ρ relaxation identifies a hidden intermediate in DNA base-pairing

Abstract

Abstract 1 H R 1ρ Relaxation dispersion (RD) NMR experiments provide valuable atomic-level insights into transient, high-energy conformational states of biomolecules. However, cross-relaxation artifacts can hamper its interpretation and therefore limiting broader adoption. This study explicitly quantifies cross-relaxation effects on 1 H R 1 ρ relaxation rates, extending the general applicability of ¹H R 1ρ to probe dynamics at natural abundance. Artifacts were found to be negligible for neighbouring dipolar-coupled protons, >3 Å apart, and a concept for identification for protons less than 3 Å is provided. This approach revealed a second excited state (ES2) in DNA base-pairing that extends the well-established Watson-Crick-Franklin (WCF) ground state (GS) – Hoogsteen (HG) equilibrium. A structural model for ES2 is proposed based on evidence from 1 H R 1ρ RD, trapping via DNA modifications, metadynamics simulations, and DFT-based chemical shift calculations. ES2 was stabilised by the anticancer drug Actinomycin D, providing direct experimental evidence that small molecule can remodel conformational landscape of DNA. Together, these results demonstrate both a methodological advance by establishing reliable conditions for 1 H R 1ρ RD studies, and a mechanistic discovery of a drug-stabilised intermediate in DNA base-pairing dynamics.