Potential G-quadruplex forming sequences (PQS) are enriched in cancer-related genes and immunoglobulin class-switch recombination. They are prevalent in the 5'UTR of transcriptionally active genes, thereby contributing to the regulation of gene expression. We and others previously demonstrated that the PQS located in the non-template strand leads to an R-loop formation followed by a G-quadruplex (G4) formation during transcription. These structural changes increase mRNA production. Here, we present how single-molecule technique was used to observe cotranscriptional G4 and R-loop formation and to examine the impact on transcription, particularly for the initiation and elongation stages.