Fig. 1.

Single-molecule fluorescence cotranscriptional folding assay. (A) Model for TPP-induced structural transition of the E. coli ThiM riboswitch. The fluorophore-labeling positions for single-molecule studies are indicated by green and red boxes (green for Cy3 or Dy547, red for Dy647). (B) Experimental scheme. Dy647-labeled seed RNA was incubated with a template DNA strand and phage T7 RNAP in a tube for 50 min at 37 °C. To assemble a full EC, Cy3-labeled UTP and a nontemplate DNA strand were added to the tube and incubated for 20 min. ECs were immobilized on a polymer-passivated quartz surface using streptavidin–biotin interactions. Elongation was resumed by injecting NTP, while RNA folding was observed using a single-molecule FRET microscope. (C) Single-molecule fluorescence images of EC (Top) and control (ctrl) images of nonspecifically bound Cy3-UTP (Bottom). The colocalized Cy3 and Cy5 spots are enclosed by circles. The percentage of acceptor spots colocalized with donor spots was estimated as 57 ± 14% from 10 measurements.