1H R1ρ 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 R1ρ relaxation rates, extending the general applicability of ¹H R1ρ 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 1H R1ρ RD, trapping via DNA modifications, metadynamics simulations, and DFT-based chemical shift calculations. ES2 was stabilised by the anticancer drug
