Determining Trap Compliances, Microsphere Size Variations, and Response Linearities in Single DNA Molecule Elasticity Measurements with Optical Tweezers

We previously introduced the use of DNA molecules for calibration of biophysical force and displacement measurements with optical tweezers. Force and length scale factors can be determined from measurements of DNA stretching. Trap compliance can be determined by fitting the data to a nonlinear DNA elasticity model, however, noise/drift/offsets in the measurement can affect the reliability of this determination. Here we demonstrate a more robust method that uses a linear approximation for DNA elasticity applied to high force range (25–45 pN) data. We show that this method can be used to assess how small variations in microsphere sizes affect DNA length measurements and demonstrate methods for correcting for these errors. We further show that these measurements can be used to check assumed linearities of system responses. Finally, we demonstrate methods combining microsphere imaging and DNA stretching to check the compliance and positioning of individual traps.

Sample preparation method to improve the efficiency of high-throughput single-molecule force spectroscopy

Inefficient sample preparation methods hinder the performance of high-throughput single-molecule force spectroscopy (H-SMFS) for viscous damping among reactants and unstable linkage. Here, we demonstrated a sample preparation method for H-SMFS systems to achieve a higher ratio of effective target molecules per sample cell by gas-phase silanization and reactant hydrophobization. Digital holographic centrifugal force microscopy (DH-CFM) was used to verify its performance. The experimental result indicated that the DNA stretching success ratio was improved from 0.89% to 13.5%. This enhanced efficiency preparation method has potential application for force-based DNA stretching experiments and other modifying procedures.

Nanochannel-Confined TAMRA-Polypyrrole Stained DNA Stretching by Varying the Ionic Strength from Micromolar to Millimolar Concentrations

Large DNA molecules have been utilized as a model system to investigate polymer physics. However, DNA visualization via intercalating dyes has generated equivocal results due to dye-induced structural deformation, particularly unwanted unwinding of the double helix. Thus, the contour length increases and the persistence length changes so unpredictably that there has been a controversy. In this paper, we used TAMRA-polypyrrole to stain single DNA molecules. Since this staining did not change the contour length of B-form DNA, we utilized TAMRA-polypyrrole stained DNA as a tool to measure the persistence length by changing the ionic strength. Then, we investigated DNA stretching in nanochannels by varying the ionic strength from 0.06 mM to 47 mM to evaluate several polymer physics theories proposed by Odijk, de Gennes and recent papers to deal with these regimes.