Parallel G-Quadruplexes Formed by Guanine-Rich Microsatellite Repeats Inhibit Human Topoisomerase I.

Area of Science:

• Biochemistry
• Molecular Biology
• Genetics

Background:

• G-rich sequences in human microsatellites can form G-quadruplex (G4) structures.
• The thermodynamic stability and folding topology of these G4s are influenced by the number of guanosines in the repeat unit.

Purpose of the Study:

• To investigate the thermodynamic stability and folding topology of G-quadruplexes formed by G-rich microsatellite sequences.
• To determine the effect of flanking sequences on G4 stability and topoisomerase I activity.

Main Methods:

• UV and Circular Dichroism (CD) spectroscopy were used to study G-quadruplex formation and stability.
• Oligonucleotides with varying G-rich repeat units (d(GGGT)4, d(GGT)4, d(GT)n) were synthesized and analyzed.
• The effect of flanking sequences on G4 stability was assessed.
• Topoisomerase I activity inhibition was measured for different G4-forming oligonucleotides.

Main Results:

• Propeller-type parallel-stranded intramolecular G-quadruplexes were formed by d(GGGT)4 and d(GGT)4.
• G4 melting temperature decreased significantly with fewer guanosines per repeat (tri-G-tetrad vs. bi-G-tetrad).
• d(GT)n-repeats did not form stable G-quadruplexes at room temperature.
• Flanking sequences destabilized parallel G4s and hindered double helix formation.
• Oligonucleotide d(CACTGG-CC-(GGGT)4-TA-CCAGTG) showed potent topoisomerase I inhibition (IC50 = 0.08 µM), linked to reduced G4 stability.

Conclusions:

• G-quadruplex stability is highly sensitive to the number of guanosines in the repeating unit.
• Flanking sequences adjacent to parallel G4s can destabilize the structure and modulate enzyme activity.
• Oligonucleotides targeting G-quadruplexes represent potential inhibitors of topoisomerase I.