1H R1{rho} Relaxation Identifies a Hidden Intermediate in DNA Base-Pairing

1H R1{rho} 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 1H R1{rho} relaxation rates, extending the general applicability of 1H R1{rho} to probe dynamics at natural abundance. Artifacts were found to be negligible for neighbouring dipolar-coupled protons, >3 [A] apart, and a concept for identification for protons less than 3[A] is provided. This approach revealed a previously hidden, 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 1H R1{rho} 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 1H R1{rho} studies, and a mechanistic discovery of a drug-stabilized intermediate in DNA base-pairing dynamics.