The persistence length of double stranded DNA determined using dark field tethered particle motion

Abstract

The wormlike chain model describes the micromechanics of semiflexible polymers by introducing the persistence length. We propose a method of measuring the persistence length of DNA in a controllable near-native environment. Using a dark field microscope, the projected positions of a gold nanoparticle undergoing constrained Brownian motion are captured. The nanoparticle is tethered to a substrate using a single double stranded DNA ͑dsDNA͒ molecule and immersed in buffer. No force is exerted on the DNA. We carried out Monte Carlo simulations of the experiment, which give insight into the micromechanics of the DNA and can be used to interpret the motion of the nanoparticle. Our simulations and experiments demonstrate that, unlike other similar experiments, the use of nanometer instead of micrometer sized particles causes particle-substrate and particle-DNA interactions to be of negligible effect on the position distribution of the particle. We also show that the persistence length of the tethering DNA can be estimated with a statistical error of 2 nm, by comparing the statistics of the projected position distribution of the nanoparticle to the Monte Carlo simulations. The persistence lengths of 45 single molecules of four different lengths of dsDNA were measured under the same environmental conditions at high salt concentration. The persistence lengths we found had a mean value of 35 nm ͑standard error of 2.8 nm͒, which compares well to previously found values using similar salt concentrations. Our method can be used to directly study the effect of the environmental conditions ͑e.g., buffer and temperature͒ on the persistence length.

Tethered particle motion mediated by scattering from gold nanoparticles and darkfield microscopy

“…We make use of 80 nm large gold particles as reporter molecules. The combination of these gold particles and darkfield microscopy results in a high SNR ob about 40 dB, which is slightly reduced by a factor of 1.2 applying an exposure time of 10 ms as reported by Brinkers et al [45]. Additionally, the small particle size minimizes the distortion of the DNA conformation by an entropic stretching force, which results from the particle's proximity to the surface of the solid support [33,45].…”

Dynamic analysis of a diffusing particle in a trapping potential

“…Few aspects of DNA dynamics have been treated before, such as the DNA dynamics in a strong stretching regime where r ∼ L [17], the DNA dynamics in response to stretching forces [18], or the dynamics of a tethered particle in the case of DNA looping [19,20] [22,23], and the statistics of Markovian processes for measuring the rate of loop formation due to protein action [19,20]. The full dynamics as we described above, however, have not been considered.…”

Mobility and height detection of particle labels in an optical evanescent wave biosensor with single-label resolution

“…A probable cause for the centred histograms is a preferred upright orientation of the analyte-antibody complex combined with a flexibility of the analyte molecule. dsDNA is known to be a semiflexible molecule with a persistence length of around 50 nm (Brinkers et al 2009, Manning 2006. A flexible analyte has a higher density of states at the centre position, and thus a higher probability to be at that position.…”