Simultaneous probing of transcription, G-quadruplex, and R-loop.

DNA and RNA can form various non-canonical secondary structures, including G-quadruplex (G4) and R-loops. These structures are considered transcriptional regulatory elements due to their enrichment at regulatory regions. During transcription, G-rich sequences in the non-template strand promote R-loop formation in the DNA template strand. These R-loops induce G4 structures in the non-template DNA strand, further stabilizing them. Additionally, the high rG: dC base-pairing within the R-loop contributes to the stability of DNA/RNA hybridization. Our previous study investigated the interplay between G4s and R-loops and its impact on transcription. We employed two techniques to demonstrate transcription-mediated G4 and R-loop formation. The single-molecule method allows us to detect intricate details of transcription initiation, elongation, and co-transcriptional R-loop and G4 formation. It provides a high-resolution view of the dynamic processes involved in transcriptional regulation. As an orthogonal approach, a gel-based assay enables the detection of the transcription-mediated R-loops and the RNA product. We can measure the progressive formation of R-loop and total RNA produced from transcription by analyzing gel electrophoresis patterns. In summary, these techniques provide valuable insights into the non-canonical nucleic acid structures and their impact on gene expression.