Extended Data Fig. 1: Measurement of p_stack by 2D aromatic [¹³C, ¹H] SOFAST-HMQC⁴⁴.

a, p_stack (see Methods) for all TAR mutants U_0-7 and wt with and without the A27U-U38A and A27-deaza-N7 base-triple disrupting mutations. b, Differences in ΔΔG_{penalty,stack} between the wt A27-U38 version of each bulge variant to its two corresponding base-triple disrupted variants, U27-A38 and N7-deaza-A27, are small (within +/−0.3 kcal/mol). This correspondence in stacking is indicated by the strong linear correlations observed between stacked populations for the wt base triple mutants versus their base triple disrupted counterparts, Pearson correlation (r) and line of best fit shown, where the colors correspond to the bulge length as shown in panel a. c, Sets of overlayed spectra for wt and all bulge mutants U_0-7. For each, the wt base triple construct is black and the base-triple disrupting mutants are overlayed, A27U-U38A in blue and A27-deaza-N7 in green. The wt spectrum is fully assigned, for the bulge constructs the stacking reporter residues A22 and U23 are indicated.