Fig. 3: Solid-state NMR reveals the presence of non-canonical base pairs or tetrads in eDNA.

A Representative solid-state 2D ¹H–¹⁵N through-space heteronuclear correlation (HETCOR) spectrum of extracellular nucleic acid (NA) gel isolate in double-distilled water (2 mg), T = 25 °C. Red ovals show the cross-peaks from direct N–H couplings of G (N1–H1) and T/U (N3–H3) when the protons are hydrogen-bonded to nucleobase nitrogen atoms (i.e. NH···N). Green ovals indicate the cross-peaks generated from indirect (via hydrogen bonds) N–H couplings between G(H1) and C(N3) as well as between T/U(H3) and A(N1). The threshold for the A(N1)–T/U(H3) correlation is increased due to lower signal intensity for that particular coupling. Cross-peaks denoted by red and green ovals indicate Watson–Crick G–C and U/T–A base pairings, respectively. Blue ovals show cross-peaks from direct N–H couplings of G (N1–H1) and T/U (N3–H3) when the protons are hydrogen-bonded to carbonyl oxygen (i.e. NH···O). These cross-peaks reveal non-canonical base pairings between G, T or U and G, T, U or C as well as the possibility of G-tetrad formations. B Canonical and non-canonical base pairing schematics illustrate the possible correlations observed in the HETCOR spectrum. The correlations are colour-coded to match the spectrum. Covalent and hydrogen bonds are indicated by solid and dashed lines, respectively.