G-Quadruplex Aptamer-Ligand Characterization.

Area of Science:

• Biochemistry and Molecular Biology
• Structural Biology
• Nucleic Acid Aptamer Research

Background:

• G-rich DNA sequences can form G-quadruplex (G4) structures, which have potential therapeutic applications.
• Understanding the structural dynamics and interactions of specific aptamers, like AT11-L2, is crucial for their development.
• Nucleolin (NCL) is a protein implicated in cancer progression and is a potential target for aptamer-based therapies.

Purpose of the Study:

• To elucidate the G-quadruplex (G4) conformation and stability of the DNA aptamer AT11-L2 under varying conditions.
• To investigate the binding interactions between AT11-L2 G4 and various G4-binding ligands.
• To determine if AT11-L2 G4 can target nucleolin (NCL) and assess its potential in NCL-positive cancer cells.

Main Methods:

• Circular dichroism, UV-Vis, and NMR spectroscopy were used to assess G4 formation and topology.
• Thermal melting (Tm) studies evaluated the stability of the AT11-L2 G4 structure.
• Fluorescence titrations were employed to quantify binding affinities (Kd) with ligands and NCL. In silico studies were performed for structural analysis.

Main Results:

• AT11-L2 forms G4 structures, with topology dependent on KCl concentration; parallel topology predominates at 100 mM KCl.
• The aptamer exhibits weak stability (Tm = 38.9 °C in 100 mM KCl), but G4 stabilization occurs with ligands 360A, BRACO-19, and PhenDC3.
• TMPyP4 disrupts the G4 structure, while AT11-L2 G4 shows high affinity (nM range) for NCL and co-localizes with it in cancer cells.

Conclusions:

• AT11-L2 forms a KCl-dependent G4 structure with moderate stability, which can be modulated by small molecule ligands.
• The aptamer demonstrates significant binding affinity for nucleolin (NCL), suggesting its potential as an NCL-targeting agent.
• AT11-L2 G4 co-localization with NCL in cancer cells supports its potential application in targeted cancer therapy.