Impact of a Snap-Back Loop on Stability and Ligand Binding to a Parallel G-Quadruplex

Area of Science:

• Molecular Biology
• Biophysics
• Structural Biology

Background:

• Genomic DNA sequences, including the MYC promoter, can form G-quadruplex structures.
• These G-quadruplexes can feature snap-back loops, influencing their properties.

Purpose of the Study:

• To compare the relative stabilities and ligand binding affinities of two G-quadruplex structures: a snap-back loop mutant (Pu22T) and the parallel MYC quadruplex.
• To investigate the structural basis for differences in ligand interaction.

Main Methods:

• Optical spectroscopy (FRET competitive melting assay)
• Microcalorimetry (Isothermal Titration Calorimetry)
• Nuclear Magnetic Resonance (NMR) spectroscopy

Main Results:

• Both Pu22T and MYC G-quadruplexes exhibit similar thermal stabilities.
• An indoloquinoline ligand binds with similar enthalpy-driven affinity to both structures, but Pu22T has fewer high-affinity sites.
• NMR footprints reveal ligand binding at the 5'-outer tetrad of Pu22T, forming a binding pocket, while the snap-back loop restricts binding at the 3'-outer tetrad.

Conclusions:

• The presence of a snap-back loop in G-quadruplexes does not significantly alter thermal stability compared to parallel quadruplexes.
• Snap-back loops impose steric constraints that modulate ligand binding site accessibility and affinity.
• Understanding these structural-functional relationships is crucial for G-quadruplex-targeted drug design.