Quantifying Microsecond Solution-Phase Conformational Dynamics of a DNA Hairpin at the Single-Molecule Level

Area of Science:

• Biophysics
• Molecular Biology
• Physical Chemistry

Background:

• Understanding biomolecular mechanisms requires quantifying conformational dynamics.
• Biomolecules exhibit heterogeneity, necessitating single-molecule methods in solution.
• Previous methods faced challenges in resolving rapid dynamics and heterogeneity.

Purpose of the Study:

• To measure microsecond conformational dynamics of DNA hairpins at the single-molecule level.
• To develop and validate a single-molecule technique for resolving kinetic heterogeneity.
• To investigate the dynamics of DNA hairpins in solution.

Main Methods:

• Utilized an anti-Brownian electrokinetic (ABEL) trap for single-molecule measurements.
• Employed two-dimensional fluorescence lifetime correlation (2DFLCS) analysis.
• Performed ABEL-2DFLCS on individual molecules to capture heterogeneity.

Main Results:

• Resolved microsecond conformational dynamics of DNA hairpins.
• Identified underlying heterogeneity in fluorescence lifetimes and kinetic parameters.
• Observed rapid (
• Successfully analyzed mixtures of DNA hairpins.

Conclusions:

• ABEL-2DFLCS accurately quantifies single-molecule dynamics and heterogeneity.
• The technique provides a more detailed view of biomolecular conformational transitions.
• This method advances the study of complex biological systems at the molecular level.