Figure 1.

(A) Watson–Crick (WC) base pairs (bps) exists in dynamic equilibrium with transient low-abundance Hoogsteen (HG) bps through flipping of the purine base 180° about the glycosidic bond from anti to syn conformation. This is also accompanied by a net reduction of C1΄–C1΄ distance by ∼2.0–2.5 Å. Shown are the average populations (p_WC/p_HG) for WC and HG bps as measured by nuclear magnetic resonance (NMR) relaxation dispersion (RD) methods (33,35). (B) m¹A stabilizes HG bp due to steric clash between the methyl group at m¹A-N1 position and T-H3. Nuclear Overhauser effect (NOE) distance connectivity between complementary nucleotides that help in distinguishing HG, WC and reverse-HG pairing modes are indicated using arrows. (C) A₂- and A₆-DNA duplexes used in this study. (D) 1D imino ¹H spectra of A₂-DNA (green), A₂-DNA^m1A16 (orange), A₆-DNA (red) and A₆-DNA^m1A16 (blue). Spectra for for A₂-DNA/A₆-DNA and A₂-DNA^m1A16/A₆-DNA^m1A16 were recorded at 25 and 9°C, respectively. Sites experiencing exchange broadening are denoted with an asterisk (*). (E) T9-H3–A16-H8, T9-H3–A-H62 and T9-H7–A-H62 NOE connectivity distinguishes the formation of HG bp from other pairing modes in A₆-DNA^m1A16 (19) (see Supplementary Figure S3 for A₂-DNA^m1A16). Sequential imino NOEs between T9-H3 with T8-H3 and G10-H1 indicate the formation of a stable duplex.