Extended Data Fig. 2. Photophysical and biophysical characterization of Squash.

a, Absorbance spectra (50 μM RNA, 5 μM DFHBI-1T) of DFHBI-1T alone and in complex with Squash demonstrates a smaller red shift (Abs max: 450 nm) compared to Spinach (Abs max: 470 nm)²⁰ and Broccoli (Abs max: 469 nm)²¹. Excess RNA was used to ensure binding of nearly all fluorophore.

b, DFHO shows a red-shifted absorbance spectrum upon binding Squash (50 μM RNA, 5 μM DFHO).

c, Both Squash and Broccoli shows similar emission maxima. Squash also showed higher fluorescence (10 μM RNA, 1 μM DFHBI-1T, ex. 452 nm for Squash and 472 nm for Broccoli) intensity with DFHBI-1T compared to DE2–6.

d, Fluorescence spectra (10 μM RNA, 1 μM DFHO, ex. 495 nm for Squash and 505 nm for Corn) showed a red-shifted Squash emission maxima (562 nm) compared to Corn (545 nm), consistent with a different mode of interaction between DFHO and Squash compared to Corn. Squash also showed more than two-fold higher fluorescence intensity with DFHO compared to Corn.

e, K_d was measured by titration of 50 nM RNA with DFHBI-1T and then fitting the data using a one-site saturation model. Data represent mean values ± s.d. for n=3 independent experiments.

f, Binding kinetics were performed as reported previously⁵⁰. The fluorescence signal trace was fitted with a monoexponential curve to extract k_obs. k_obs was plotted as a function of total RNA (50 nM RNA) and fluorophore concentration. k_on and k_off were extracted as the slope and intercept, respectively. Squash k_off (0.014 ± 0.008 s⁻¹) is very similar to that of Corn (0.018 ± 0.002 s⁻¹). k_on for Squash-DFHO (162300 ± 8100 M⁻¹ s⁻¹) is seven-times higher than Corn-DFHO (23000 ± 3000 M⁻¹ s⁻¹) which could be due to the high folding of Squash.

g,h, K_d measurements for Broccoli and Squash binding to BI (g) and DFHO (h) were performed as in panel e, using 50 nM RNA and then fitting the data using a one-site saturation model. Data represent mean values ± s.d. for n=3 independent experiments.