Probing dynamics of individual protein molecules on DNA using a nanopore

Dr. Yanina Cesa (BPE group)

Objective

The challenge in this project is to measure with nanometer precision the position and movement of individual protein molecules as their bound to or as they move towards their specific binding site on a single DNA molecule.

Methodology

Pulling the DNA-protein complexes with an electric field to and through a nanopore, one at a time, the position of the bound protein with respect to the DNA molecule can be obtained measuring the current through the nanopore. This method is able to measure in a short time, allowing for fast screening of preferential protein binding sites on specific DNA fragments. Direct assessment of the protein movement along the DNA can be realized by probing the location of individual protein molecules on the same DNA molecule continuously. Combining the positioning and the movement information, we will provide insight in the search mechanisms that protein molecules use to find the specific binding site on the DNA molecule.

The first step towards our final objective is to develop the technique that will allow us to detect whether a protein is bound to a specific DNA sequence and to localize it with high resolution. The nanopore (slightly larger than the protein-DNA complex cross section) separates two compartments filled with ion solutions. Applying a voltage across the nanopore an ion flow will be induced. We can measure it directly as the electrical current flowing between the electrodes. When DNA or DNA-protein complex is added to the compartment where the negative electrode is placed, the negatively charged molecules are pulled, one at a time, to and through the nanopore. The presence of the molecule inside the hole results in a blockage of the ion flow measurable as a decrease in the current during the translocation time. The step in the current will be related to the cross section of the molecule going through the pore, then a larger reduction in the current (respect to the free pore level) will be observed when the protein bound to the DNA is traversing the pore. Measuring accurately (nanometer precision) the onset on this reduction the positioning of the protein with respect to the DNA molecule will be possible.