Single-molecule TPM studies on the conversion of human telomeric DNA

Area of Science:

• Biophysics
• Molecular Biology
• Genetics

Background:

• Human telomeres feature G-rich DNA sequences prone to forming G-quadruplexes (G4s).
• G4 formation is influenced by monovalent cations like sodium (Na+) and potassium (K+).
• Understanding G4 dynamics is crucial for telomere stability and potential therapeutic targeting.

Purpose of the Study:

• To investigate the unfolding process and conformational changes of human telomeric G-quadruplexes (G4s) at the single-molecule level.
• To elucidate the role of monovalent cations and antisense sequences in G4 stability and structural transitions.
• To determine the pathway of spectral conversion between Na+- and K+-folded G4 states.

Main Methods:

• Development and application of a single-molecule tethered particle motion (TPM) assay.
• Real-time monitoring of DNA tether length changes to detect G4 formation and unfolding.
• Titration experiments with varying salt concentrations and addition of antisense sequences.

Main Results:

• Observed distinct unfolding time constants (82 s and 3152 s) in the presence of antisense sequences.
• Demonstrated strong dependence of G4 folding equilibrium constant on Na+ concentration (1.75 at 5 mM to 3.40 at 15 mM).
• Found no evidence of a fully unfolded intermediate during the conversion from Na+-folded to K+-folded G4 states, suggesting loop rearrangement.

Conclusions:

• Single-molecule TPM is effective for studying DNA conformational changes.
• G4s in human telomeric sequences likely interconvert between folded states via loop rearrangement rather than complete unfolding.
• The pathway of G4 structural conversion is cation-dependent and may not involve a fully unfolded intermediate on minute timescales.