Measurements of smFRET

smFRET measurements were performed using a home-built total internal reflection fluorescence (TIRF) microscope (IX71, Olympus) equipped with a 100× objective (UAPON 100XOTIRF, Olympus), a 532-nm laser (CL532-075-L, CrystaLaer) and an EMCCD (iXon DU-897D, Andor). Details of the microscope setup are described in Supplementary Methods.

Dye-labeled RNA complexes were immobilized on a slide surface, in a buffer containing 40 mM HEPES-KOH (pH 7.5), 70 mM NH₄Cl, 7 mM Mg(OAc)₂ and 1.7 mM Trolox (Sigma-Aldrich). To improve dye stability, the following oxygen scavenging system (43) was also included in the buffer: 2.6 mM protocatechuic acid (PCA; Sigma-Aldrich) and 48 nM protocatechuate 3,4-dioxygenase (PCD; Sigma-Aldrich). Fluorescence movies were recorded at 20 Hz using SMET, a LabView-based program package (44), and processed by the IDL (Exelis) scripts released from Taekjip Ha's lab (https://cplc.illinois.edu/software). Time-evolved smFRET traces were analyzed by custom-written MATLAB programs. The first 10 data points from each trace were averaged and reported for FRET distribution.