Heidelinde R. C. Dietrich

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

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 (dsDNA) 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 nm), 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 temperature) on the persistence length.

HU Protein Induces Incoherent DNA Persistence Length

HU is a highly conserved protein that is believed to play an important role in the architecture and dynamic compaction of bacterial DNA. Its ability to control DNA bending is crucial for functions such as transcription and replication. The effects of HU on the DNA structure have been studied so far mainly by single molecule methods that require us to apply stretching forces on the DNA and therefore may perturb the DNA-protein interaction. To overcome this hurdle, we study the effect of HU on the DNA structure without applying external forces by using an improved tethered particle motion method. By combining the results with DNA curvature analysis from atomic force microscopy measurements we find that the DNA consists of two different curvature distributions and the measured persistence length is determined by their interplay. As a result, the effective persistence length adopts a bimodal property that depends primarily on the HU concentration. The results can be explained according to a recently suggested model that distinguishes single protein binding from cooperative protein binding.

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

We measured light scattered from gold nanoparticles with darkfield microscopy in order to perform single molecule detection based on tethered particle motion (TPM). This combination results in a signal to noise ratio of about 40 dB, which allowed us to use 80 nm diameter gold particles as reporters instead of the typically used polystyrene particles whose sizes are up to 1 µm. The particle size is crucial in TPM experiments as it can induce a volume-exclusion effect, which results in a stretching force acting on the DNA tether. This affects both the biophysical and statistical properties of the anchored DNA and hence the interpretation of the experimental data. We demonstrated that the gold nanoparticles and darkfield microscopy can be used to characterize the confined Brownian motion of dsDNA-tethered gold particles with a spatial precision of 3 nm. Physical parameters such as the spring constant of the tethered DNA fragment and the persistence length can be derived from the two dimensional (2D) (x, y) projected image data. We have applied this method to various MgCl2 and glycerol concentrations as a proof of principle.

Single molecule detection of tuberculosis nucleic acid using dark field Tethered Particle Motion

Current methods for tuberculosis nucleic acid detection require amplification and labeling before detection is possible. We propose here a method for direct detection using Tethered Particle Motion: gold nanoparticles are tethered to a glass substrate by single-stranded DNA molecules consisting of the complementary sequence to the target. Detection takes place by observing a change in the motion of the nanoparticles. The particles are imaged by a dark field microscope and captured on an EMCCD camera. Single particle tracking is carried out through maximum likelihood estimation of the Poisson noise limited Gaussian image profile using a parallelized algorithm on a GPU. The method is characterized by biophysical modeling and the ability to detect nucleic acids is shown. This single molecule method is suitable for multiplexing and could form the basis of an exquisitely sensitive method of detecting the presence of nucleic acids derived from human pathogens directly from patient material.

Studies of Single Molecules in their Natural Form

Single molecule studies make possible the characterization of molecu- lar processes and the identification of biophysical sub-populations that are not acces - sible through ensemble studies. We describe tethered particle motion, a method that allows one to study single molecules in their natural form without having to apply any external forces. The method combines darkfield microscopy with a metal nano- bead. It permits the study of the biophysical properties of the tethered particles, as well as protein-DNA interactions. The method is not suitable for in vivo studies, and we therefore describe two other methods that are appropriate for live-cell imaging.

Gold nanoparticles: a novel application of spectral imaging in proteomics -- preliminary results

The intense research in proteomics is demanding for fast, reliable and easy-to-use methods in order to study the proteome. In this proceeding we report the development of such a novel research tool based on spectral imaging and Resonance Light Scattering gold particles. This method will allow the study of DNA-protein interactions. We suggest a broad range of applications: the screening of proteins binding to a specific DNA sequence, the analysis of binding affinities between protein and DNA, and the investigation of the influence of environmental conditions on the binding. We will explain the principle, first experiments and first results based on Brownian motion.

A new optical method for characterizing single molecule interactions based on dark field microscopy

Single-molecule techniques continue to gain in popularity in research disciplines such as the study of intermolecular interactions. These techniques provide information that otherwise would be lost by using bulk measurements that deal with a large number of molecules. We describe in this report the motion of tethered DNA molecules that have been tagged with gold nanobeads and observed under dark field microscopy to study single molecular interactions (SMI). We further report on the derivation and use of several physical parameters and how these parameters change under differing experimental conditions.

Adaptation of nanoarrays for the study of α-synuclein aggregation: preliminary results

In previous publications we have shown that we can perform enzymatic reactions in nanoarrays by means of a microarray-reader based on a conventional microscope. In this publication we report on a modification of this system in order to monitor the aggregation kinetics of the natively unfolded protein α-synuclein. We describe the motivation for this development, the problems associated with the miniaturization of the aggregation assay, and the validation of our modifications.