Fig. 5: Single-molecule investigation of DNA structural changes and transcription dynamics affected by DNA superhelicity.

a Schematic illustration of [FRET1] designed for direct observation of elongating RNAP and DNA structural changes. PQS is positioned 30 bp downstream of TSS in the non-template strand (NT-sp30), with Cy3 located 27 bp (+ 27) and Cy5 located 47 bp (+ 47) downstream from TSS. Linearized, relaxed, and supercoiled [FRET1] constructs were generated to investigate the impact of DNA superhelicity on RNAP movement and DNA structural modifications. In the absence of any event, the construct exhibits an expected FRET value (E) of ~ 0.3. As elongating RNAP approaches Cy3, the E remains at ~ 0.3, while the Cy3 signal is enhanced due to Protein-Induced Fluorescence Enhancement (PIFE). The expected E of R-loop and G4 is ~ 0.7 and ~ 0.9, respectively. b, c Representative single-molecule time traces of RNAP transcription for relaxed (b) and supercoiled (c) [FRET1] DNA constructs. The top graph shows Cy3 (green) and Cy5 (red) signals; the middle graph shows the combined intensity of Cy3 and Cy5, illustrating PIFE occurrence by RNAP passing Cy3; the bottom graph shows FRET changes, indicative of DNA structural alterations. The dashed gray line marks the time point when RNAP is introduced into the channel. b With the relaxed [FRET1], no FRET changes are observed, but short-lived PIFE signals are present. c With the supercoiled [FRET1], FRET transitions between ~ 0.3 and ~ 0.7 occur with short-lived PIFE peaks (d) A closer examination of the dashed black box on the left side of (c) reveals a distinct temporal shift between PIFE and FRET peaks, with a FRET peak emerging shortly after a PIFE peak. e The dashed black box on the right side of (c) shows that after several transitions, an additional FRET change from ~ 0.6 to ~ 0.9 is observed. f FRET histogram of supercoiled [FRET1] after 30 min transcription (first from the top) and subsequent RNase H treatment (second). The orange boxed area corresponds to R-loop structures, which disappear in the presence of RNase H. The blue boxed area corresponds to stable G4 structures, which remain unaffected by RNase H treatment. FRET histogram of supercoiled [FRET1] after 30 min transcription in G4 destabilization condition (third from the top) and subsequent RNase H treatment (forth). The fraction of red boxed area decreases, corresponding to G4 destabilization and the blue box disappears in RNase H treatment, further confirming the state of R-loop and G4. g The quantification of PIFE peaks before forming stable G4 (E ~ 0.9) for linearized, relaxed, and supercoiled [FRET1] with more than 200 molecules for each experiment. See details in the method section. The exact data and P-values are provided in the Source data file. ***P  <  0.0005, NS: nonsignificant (two-sided unpaired t test) (h) Time course of G4/R-loop formation affected by DNA superhelicity and RNAP concentration. The impact of DNA superhelicity on G4/R-loop formation rate in the following order: supercoiled [FRET1] with 1X RNAP (circle) > relaxed [FRET1] with 5X RNAP (triangle) > relaxed [FRET1] with 1X RNAP (triangle) > linearized [FRET1] with 5X RNAP (square).