Fig. 3: Chili binds the phenol form of DMHBO⁺ via H-bonding to N7 of G15.

a The phenol/phenolate equilibrium of DMHBO⁺ allows monitoring of RNA folding. b Temperature-dependent absorbance spectra of the parent Chili RNA–DMHBO⁺ complex (10 µM) and white light images of 100 µM solutions upon cooling from 85 °C to room temperature. c Absorbance (A) at 450 nm (yellow) and 550 nm (purple) as a function of temperature. The inflection point at 70 °C fits well to the reversible thermal melting transition of the RNA monitored at 260 nm (right axis: hyperchromicity (hyp₂₆₀), gray). d H-bonding between the ligand and N7 of G15 in wt-Chili and selected mutations. e c⁷G15-modified Chili RNA is not able to shift the phenol/phenolate equilibrium. Absorbance spectra of the parent complex (solid black line) in comparison to free ligand (dashed black line) and with c⁷G RNA mutant (solid red line). f Absorbance and g fluorescence spectra of Chili–DMHBO⁺ complexes containing point mutations. The C40U mutation (Wobble base pair) is tolerated, while G15A completely disrupts ligand binding and fluorescence. Compensatory mutations of G15:C40 to A15:U40 only partially restore the fluorogenic binding site and result in a blue-shifted absorption maximum. RNA and ligand concentrations are 10 µM in b and e and 5 µM in f and g.