Stabilization of a DNA aptamer by ligand binding

Area of Science:

• Biochemistry and Molecular Biology
• Nucleic Acid Chemistry
• Cancer Therapeutics

Background:

• G-rich aptamers offer advantages over monoclonal antibodies for therapeutic applications.
• AT11-B1 is a modified G-rich aptamer derived from AS1411, with a specific sequence modification.
• G-quadruplex (G4) structures are key targets for aptamer-based therapies.

Purpose of the Study:

• To analyze the G-quadruplex formation and stabilization of the AT11-B1 aptamer.
• To investigate the binding interactions of various ligands with the AT11-B1 G4 structure.
• To explore the interaction of AT11-B1 G4 with nucleolin (NCL) and its cellular uptake in cancer cells.

Main Methods:

• Circular dichroism (CD) spectroscopy to determine G4 topology and thermal stability.
• Förster resonance energy transfer (FRET-melting) assays for G4 stabilization studies.
• Nuclear magnetic resonance (NMR) spectroscopy and in silico studies to elucidate ligand binding modes and G4 structure.
• Molecular dynamics simulations to analyze aptamer-protein interactions.
• Cellular uptake studies in a NCL-positive cancer cell line.

Main Results:

• AT11-B1 predominantly forms a parallel G4 structure in the presence of KCl or ligands.
• PhenDC3 significantly stabilized the AT11-B1 G4 structure, with a ΔT_m of ≥30 °C.
• Ligands exhibited high affinity for AT11-B1 G4, with NMR revealing four G-tetrad layers.
• In silico analysis indicated ligand binding primarily through stacking interactions.
• Molecular dynamics simulations showed AT11-B1 G4 interacting with nucleolin's RNA binding domain 2 and linker region.
• AT11-B1 G4 was successfully internalized into NCL-positive tongue squamous cell carcinoma cells.

Conclusions:

• The study characterizes the AT11-B1 G4 structure and its stabilization by small molecule ligands.
• Ligand binding and stabilization of AT11-B1 G4 are confirmed, offering insights for scaffold identification.
• The interaction with nucleolin and cellular internalization highlight AT11-B1's potential as a cancer-targeting agent.
• These findings support the development of AT11-B1-based therapeutics for cancers overexpressing nucleolin.