Research assistant Sanjin Marion published a paper titled “Relevance of the drag force during controlled translocation of a DNA-protein complex through a glass nanocapillary” published in Nano Letters (IF=13.6 in 2014.) together with UKF project collaborators prof. A. Rađenović and R. Bulushev from the Ecole Polytechnique Federale Lausanne. The work presents results of controlled translocation of individual DNA bound proteins with optical tweezers. S. Marion contributed to the article through interpretation and theoretical modeling of experimental results.

Relevance of the drag force during controlled translocation of a DNA-protein complex through a glass nanocapillary

Roman D. Bulushev, Sanjin Marion, Aleksandra Radenović

Nano Letters 15, 7118-7125 (2015). doi: 10.1021/acs.nanolett.5b03264

 

Nanocappilaries, nanopores and similar objects are becoming an interesting tool for single molecule experiments with special emphasis on future applications towards cheap and fast DNA sequencing. The work described in this paper represents a first step towards experimental localization of binding sites for different proteins on DNA by nanopore/nanocapillary methods.

In the experiment DNA was bound to specially prepared micrometer-sized beads and put in a solution with DNA binding proteins. A single DNA molecule was forced to enter a capillary (with an opening of 20-100 nm) by an external electrical field while simultaneously being held in position with the force exerted by an optical tweezer on the bead. This enabled the authors to do repeated controlled translocations of the DNA molecule through the capillary, with or without bound proteins and in physiological conditions, all the while noting a change in both the applied force and conductivity of the capillary.

This method allowed the characterization of a protein bound to DNA by a simultaneous change of the force and ionic current signal with respect to the bare DNA experiments. Controlled displacement of the protein away from the nanocapillary opening revealed decay in the values of the force and ionic current. Negatively charged proteins EcoRI, RecA, and RNA polymerase formed complexes with DNA that experienced electrophoretic force lower than the bare DNA inside nanocapillaries.

Interestingly, force profiles obtained for DNA-RecA were different than those previously obtained using nanopores in membranes and optical tweezers. To explain the differences, translocation experiments were modeled as a stochastic process which included the state of the DNA and bead. Different contributions to the total free energy of the protein were included, such as electrostatics, DNA entropy and bending and electroosmotic flow. This enabled the authors to identify an unexpectedly large contribution coming from the electroosmotic flow in the effective (measured) charge densities of single and fiber proteins. Such behavior implies the drag force as the dominant influence on a DNA-protein complex inside nanocapillaries with respect to electrostatic contributions. The drag force originates from charge accumulation around the surface of the quartz capillaries and charged DNA and proteins. Under an external electrical field a local fluid flow is induced due to a local force on this accumulated charges. The resulting drag force is Stokes like and changes the measured protein charge.