RPA-mediated unfolding of systematically varying G-quadruplex structures

Area of Science:

• Molecular Biology
• Biophysics
• Genetics

Background:

• G-quadruplexes (GQ) are noncanonical DNA structures formed by guanine-rich sequences.
• GQ structures can impede essential DNA metabolic processes like replication and repair.
• Replication protein A (RPA) is a key protein known to destabilize GQs.

Purpose of the Study:

• To investigate the unfolding of G-quadruplex structures mediated by replication protein A (RPA).
• To analyze how loop length and the number of G-tetrad layers influence GQ stability against RPA.
• To elucidate the mechanism and kinetics of RPA-mediated GQ unfolding.

Main Methods:

• Single-molecule Förster Resonance Energy Transfer (smFRET) was employed to study GQ unfolding.
• Two distinct groups of GQ structures with varying loop lengths and G-tetrad layer numbers were analyzed.
• Steady-state stability and transition times for RPA-mediated unfolding were measured.

Main Results:

• GQ stability against RPA unfolding increased linearly with more G-tetrad layers and shorter loop lengths.
• GQ stability varied over three orders of magnitude across different constructs.
• Shorter loops (<3 nucleotides) or more layers (>3) resulted in significant folded populations at physiological RPA concentrations (≈1 μM).
• The transition time for RPA-mediated GQ unfolding was consistently measured at 0.35 ± 0.10 s for all constructs.

Conclusions:

• GQ structures with specific loop lengths and layer numbers exhibit significant stability against RPA.
• The observed stability suggests that certain G-quadruplex structures may be physiologically viable.
• A two-step mechanism is proposed for RPA-mediated G-quadruplex unfolding, consistent with experimental observations.