Extended Data Fig. 1: Secondary-structure prediction using MC-Fold.

Secondary-structure rearrangements among the ten lowest-energy structures were calculated using MC-Fold²¹. Ranking (numbers in parentheses) according to the predicted energy difference, based on the minimum free energy (MFE), is indicated in each label (ΔΔG(n) in units of unreferenced kcal mol⁻¹, as described²¹). Secondary structures with a single base-pair opening in the cUUCGg region are omitted. a, The miR-34a–mSirt1 duplex connected by a cUUCGg loop (black). The MFE corresponds to a 7-mer–A1 binding site. Suboptimal structures (3) and (5) suggest possible modulation of the binding site to a 8-mer–GU and an 6-mer–A1 configuration, respectively. b, miR-34a–mSirt1 bulge construct, comprising a cUUCGg loop and a closing stem (black). The secondary-structure distribution of the miR-34a–mSirt1 bulge follows the same trend as the full-length duplex; dashed lines connect identical bulge structures. Suboptimal structures were used to validate or reject models of excited-state (ES) secondary structures on the basis of R_1ρ NMR relaxation-dispersion data. Structure (1), with the MFE, corresponds to the assigned ground-state structure (GS). Structure (3) satisfies the ¹H₁ and ¹⁵N₁ R_1ρ NMR relaxation-dispersion data on gG6(G24), being G:U base paired with tU20(U9). Structure (5) is mutually exclusive with (3) in structural terms and satisfies the ¹³C R_1ρ NMR relaxation-dispersion data measured on tA19 that indicate this residue adopting a base-paired conformation. Therefore we propose structure (3) as ES1 and structure (5) as ES2. Conformations (6) and (7) do not agree, and partially clash, with our R_1ρ NMR relaxation-dispersion data and can therefore be excluded as excited states. c, miR-34a–mSirt1 trapped excited-state duplex connected by a cUUCGg loop (black). Substituted nucleotides used to trap the excited state are in yellow. The MFE corresponds to a 8-mer binding site. d, miR-34a–mSirt1 (turquoise) trapped excited-state construct comprising an cUUCGg loop and a closing stem (black). Substituted nucleotides used to trap the excited state are in yellow. The secondary-structure distribution of the miR-34a–mSirt1 trapped excited state follows a similar trend as the full-length duplex; dashed lines connect identical bulge structures.