Correlated optical tweezers and fluorescence imaging of DNA mechanics
Duration
March-April 2026
Institution
Northeastern University Department of Physics
Tags
#
Biophysics
Research Question
The objective was to investigate the structural dynamics of lambda-phage DNA under mechanical stress. The project aimed to induce and quantify the phase transition of DNA from a double-stranded (dsDNA) to a single-stranded (ssDNA) state at a critical force of approximately 60 pN.
Methods & Instrumentation
Operated a LUMICKS Super C-TRAP system, integrating a 1064 nm dual-trap optical tweezer setup with 488 nm confocal fluorescence imaging
Tethered single DNA molecules between streptavidin-coated polystyrene beads within a microfluidic laminar flow cell
Manipulated the DNA tethers with the optcal traps and measured the applied mechanical forces by tracking laser deflection using position-sensitive diodes
Applied intercalating dyes (TO-PRO-1) and selective replication proteins (RPA488) to simultaneously visualize dsDNA and ssDNA regions during the overstretching process
Analyzed diffraction-limited confocal scans by applying full-width at half-maximum (FWHM) corrections to account for optical blurring and accurately measure molecular extension
Compared the remaining fraction of dsDNA calculated from fluorescence imagery against theoretical polymer physics models, specifically the Extensible Worm-Like Chain and Freely Jointed Chain models
Results & Significance
Successfully mapped the mechanical overstretching of the DNA directly to base-pair melting at the single-molecule level
Achieved a highly accurate 1:1 correlation between the physical extension data and the fluorescence measurements, valudated by a zero-intercept linear regression with an R-squared value of 0.9943
Identified specific melting pathways, noting that while end-peeling dominated the transition, DNA backbone nicking introduced distinct internal melting sites