Validation of G-quadruplex detection in live cells with a small G4-probe

G-quadruplexes (G4s) are secondary structures adopted by guanine-rich single-stranded DNA/RNA sequences. In the human genome, they play a relevant role in telomere metabolism, genome transcription, replication, and translation. Their presence in fixed cells was confirmed by immunofluorescence analysis thanks to the use of antibodies that specifically bind these structures. Due to their relevance in the cellular biological processes and tumor development, scientists are developing systems to map them on the human genome. To date the anti-G4 monoclonal antibody BG4 was used in different genome-wide sequencing-based techniques to map and identify the genomic regions that are actually folded in G4 in cells. Among them, the CUT&Tag is the most recent and advanced mapping technique, the advantage of which is that it allows identification of the target structures in live cells. We here investigated the possible use of a novel smaller G4-probe (G4P) for the detection of G4s in live cells by different techniques, including the CUT&Tag assay. The probe is inserted in a mammalian expression vector that allows its expression and the consequent binding to DNA G4 structures in living cells. The expression of the G4P is under the same promoter of a EGFP so that the transfection efficiency can be easily evaluated. The transfection protocol was set up in the A549 cell line and G4P and EGFP expression as well as their localization were assessed via western blot and immunofluorescence analysis. The CUT&Tag assay was set up first with positive and negative controls; next cells were transfected with the G4P expressing plasmid, filtrated, and sorted to select only cells expressing the G4P. Unfortunately, these treatments exceedingly stressed the cells and the CUT&Tag assay, which needs live and healthy cells, did not work. While the results did not validate the use of the G4P in these conditions instead of BG4 in the CUT&Tag assay, they still support its possible utility to detect G4s in live cells.