Single-molecule FRET measures bends and kinks in DNA

Area of Science:

• Biophysics
• Structural Biology
• Molecular Biophysics

Background:

• Determining precise 3D DNA structures is crucial for understanding biological functions.
• Fluorophore-linker mobility complicates distance measurements in single-molecule Förster Resonance Energy Transfer (smFRET).
• Existing methods struggle to accurately model the dynamic nature of dyes attached to DNA.

Purpose of the Study:

• To develop and validate a single-molecule FRET approach for deriving full 3D DNA structures using absolute distances.
• To investigate and account for fluorophore dynamics to improve FRET-based structural analysis.
• To enable the study of protein- or damage-induced DNA bending with high resolution.

Main Methods:

• Systematic variation of donor-acceptor distances in DNA double-helices (2-10 nm).
• Analysis of dye-dye quenching and fluorescence anisotropy to characterize fluorophore dynamics (positional and orientational).
• Application of a nonlinear conversion function (informed by MD simulations) to calculate absolute FRET distances, incorporating fluorophore dynamics.
• Quantitative statistical analysis using triangulation for conformational searching, leveraging known nucleic acid helical features.

Main Results:

• Characterization of slow positional and fast orientational fluorophore dynamics, leading to an averaged FRET efficiency.
• Successful derivation of absolute FRET distances by accounting for fluorophore mobility.
• Detection of sequence-dependent DNA bending (16 degrees) and quantification of kink angles in DNA with bulged adenosines (32° ± 6°, 56° ± 4°, 73° ± 2° for 1, 3, and 5 bases, respectively).
• Detailed description of relative helix orientation through calculation of rotation angles and shifts.

Conclusions:

• The developed single-molecule FRET method provides high accuracy for 3D DNA structure determination.
• This approach effectively addresses challenges posed by fluorophore-linker mobility, enabling precise distance measurements.
• The technique offers significant potential for studying DNA structural dynamics, including sequence-dependent bending and alterations induced by bulges or external factors.