Fig. 4: Single-Molecule Detection Platform for Observing DNA Structural Changes and RNA Polymerase Movement on Immobilized Linearized, Relaxed, or Highly Negatively Coiled 3.7 kb DNA.

a The schematic representation of immobilized 3.7 kb DNA with linearized, relaxed, or highly negatively coiled DNA templates, which are specifically labeled with Cy3 and Cy5. Each DNA construct was immobilized on a PEG-coated glass slide via biotin-neutravidin interaction for single-molecule observation. To simultaneously observe the structural configuration of G4 and R-loop, and RNAP movement, Cy3 (+ 27) and Cy5 (+ 47) were positioned across the PQS [FRET1]. To monitor the transcription initiation process, Cy3 (− 4) and Cy5 (+ 19) were positioned close to the promoter [FRET2]. b Using a nicking enzyme-based internal labeling method, Cy3, Cy5, and biotin-labeled oligomers were annealed with the 3.7 kb-sized vector and sealed through ligation. During ligation, the presence or absence of EtBr determined whether the construct was relaxed or supercoiled. See the method section for details. The reconstituted constructs were confirmed with 1% agarose gel electrophoresis. The post-stained image with SYBR Green II displays a plasmid, the original template purified from E. coli (lane 1), along with Cy3 and Cy5 labeled relaxed (lane 2) and supercoiled DNA (lane 3). The fluorescence image without post-staining (lanes 5 and 6) displays Cy3 and Cy5 labeled relaxed DNA (lane 5) and supercoiled DNA (lane 6). c Histograms of relaxed and supercoiled [FRET1] DNA measured by single-molecule TIRF, displaying ~0.3 FRET value as expected.