Extended Data Fig. 3: MCSF analysis of the miR-34a–mSIRT1 bulge and trapped excited state, and analysis of  ¹³C tA22C8 outliers.

a, b, We used the MCSF approach²² to cross-validate our candidate excited state (ES1), modelled using R_1ρ-derived ground-state-to-excited-state chemical-shift differences (a, ¹³C R_1ρ Δω data, blue dots; b, left). p_ES refers to the excited-state population (pop_ES in the main text). We also generated an ES1 mimic (trapped ES1) using a two-point substitution, predicted to stabilize the proposed conformation (b, bottom). For each reporter atom, we compared ¹³C R_1ρ Δω data with the chemical-shift differences derived from the assignment of the bulge and the trapped ES constructs (a, ¹³C Δω trapped ES (tES) data, turquoise dots). In a, The MCSF analysis validates our ES1 model (green shading), with exceptions arising from the limitations of the mimic (orange shading) and from the presence (violet shading) of a second ES (ES2, b, right). Errors for R_1ρ relaxation-dispersion-derived Δω represent 1 s.d. from fitting (see also Supplementary Methods). In b, the proposed model for ES2 satisfies the ¹³C R_1ρ Δω data measured for tA19 and gG6. GS, ground state. c, The free-energy landscape for the entire star-like three-state exchange process. (The MCSF analysis and ES2 are discussed further in the Supplementary Information, Discussion 5.) The transition coeffient (κ), is assumed to be 1 (ref. ²³), so the transition-state energies (TS1 and TS2), calculated using Supplementary equation (11), must be considered an upper limit of this exchange process. d, e, The substitution site (tU21 to tC21) perturbs the chemical environment of tA22C8 that is directly neighbouring the substituted nucleobase (orange sphere in e). Conversely, tA22C2 (green sphere), pointing towards the miR-34a strand (red), experiences an equivalent chemical environment in the bulge (blue) and trapped ES (turquoise) constructs. This explains the inconsistency in the MCSF profile for tA22C8 (Supplementary Fig. 12a, orange box). d, Secondary structure environment of tA22 in the miR-34a–mSirt1 bulge excited state (left) and trapped ES (right) constructs. The substitution site (tU21 to tC21) is highlighted. e, Overlay of average structures of the bulge ES (blue) and trapped ES (turquoise) from REMD ensembles, aligned according to residues gU7 and tA22. Residues gU7, gG8, tU21 and tA22 are shown. tA22C8 and tA22C2 ¹³C atoms are in orange and green respectively.