Reporter System for Detection of G-Quadruplexes in the Human Telomerase Reverse Transcriptase Gene Promoter Region

In 80-100% of cases, transformation of human somatic cells into tumor cells is associated with the increased expression of the catalytic subunit of telomerase reverse transcriptase (hTERT). The hTERT gene transcription inhibition in tumor cells may become one of the approaches to antitumor therapy. The hTERT promoter contains a G-rich region with length of 68 nucleotides, which is capable of forming G-quadruplexes (G4) under certain conditions in vitro. It is known that G4s interfere with activity of the human RNA polymerases. Thus, the G4 structure stabilization in the promoter could be considered as a possible strategy to reduce hTERT expression. To prove G4 formation in the hTERT promoter G-rich sequence in the double-stranded supercoiling DNA, plasmid constructs based on the pRFPCER plasmid were obtained. The plasmids contained genes of fluorescent proteins (RFP and Cerulean) and sequence of the central G4 in the hTERT promoter region. G4 formation in the central hTERT promoter region in the obtained constructs was demonstrated with the DNA polymerase stop assay. The influence of G228A and G250A substitutions on G4 stability under physiological conditions was investigated. It was established that the low-molecular weight ligands BRACO19 and TMPyP4, the well-studied stabilizers of the G4 structure, can effectively interact with the hTERT promotor central G4 in the range of concentrations 5-25 μM.