Cary 500 uv vis nir spectrophotometer

Absorbance spectra were collected using a Cary 500 UV-VIS-NIR spectrophotometer and buffer subtracted. The quantum yield (Q) of probes was determined by comparison with the published Q of ATTO 590 (0.80, Atto tec) or Cy5 (0.28, Lumiprobe). All experiments were done in RNA buffer (100 mM KCl, 10 mM NaCl, 1 mM MgCl₂, 50 mM HEPES, pH 8.0). First, absorbance was determined at the excitation wavelength (Supplementary Table 11) for a dilution series of free fluorophore and Cbl-fluorophore probes +/− different RNAs. To ensure saturation of binding, the concentration was chosen such that the final concentration of RNA was above 5 μM in the most diluted sample. The fluorescence spectrum at the emission range (Supplementary Table 11) for each sample was then recorded using a PTI-fluorimeter (1 nm steps, 1 s integration time, 2 nm slits for the excitation and 4 nm slits for detector). After subtracting the buffer background signal, the fluorescence signal for each sample was integrated and plotted vs. the absorption. The steepness of the resulting linear plot for each dilution series reports on Q relative to the reference of the free fluorophore (Supplementary Table 8). These measurements were done once for the Q determination, and spectra were comparable with absorbance/fluorescence measurements done for fold fluorescence measurements.