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

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Increased human telomerase reverse transcriptase (hTERT) expression is linked to tumor cell transformation.
• Inhibiting hTERT transcription is a potential antitumor therapy approach.
• The hTERT promoter contains a G-rich region capable of forming G-quadruplexes (G4), which can impede RNA polymerase activity.

Purpose of the Study:

• To investigate the formation of G4 structures within the hTERT promoter.
• To assess the potential of G4 stabilization as a strategy for hTERT gene transcription inhibition.
• To evaluate the efficacy of specific ligands in stabilizing hTERT promoter G4 structures.

Main Methods:

• Plasmid constructs containing the hTERT promoter G-rich sequence and fluorescent reporter genes were created.
• DNA polymerase stop assays were employed to confirm G4 formation in the hTERT promoter region.
• The impact of specific nucleotide substitutions (G228A, G250A) on G4 stability was examined.
• Interactions between G4 structures and known stabilizers (BRACO19, TMPyP4) were analyzed.

Main Results:

• G4 formation was successfully demonstrated in the central G-rich region of the hTERT promoter within plasmid constructs.
• The study investigated the influence of G228A and G250A substitutions on G4 stability.
• Low-molecular-weight ligands, BRACO19 and TMPyP4, effectively interacted with the hTERT promoter G4 at concentrations of 5-25 μM.

Conclusions:

• G4 structures can form in the hTERT promoter region, presenting a target for therapeutic intervention.
• Stabilization of these G4 structures by ligands like BRACO19 and TMPyP4 offers a promising strategy to inhibit hTERT expression.
• This research supports the development of novel antitumor therapies targeting hTERT via G4 structure modulation.