Bionanopores for the analysis of DNA-protein complexes

Introduction

At this moment small nanopores ranging in size from about 2 to 10 nm are being used for determination of the length of individual DNA molecules (i.e. DNA sizing). The measurement principle is based on the Coulter counter, a reduction in conductance as the molecules moves through the pore. Having a constant voltage across the pore, results in a reduction of the current, as a direct result of effective blocking.

(left) Schematic representation of two chambers separated by the nanopore and the electrode configuration used to actively pull DNA molecules through the pore.

(right) Example of the reduction of current as a result of the passage of a DNA-protein complex

These nanopores can be produced in a silicon-based (Si₃N₄, SiO₂) using nanotechnology tools (i.e. focused ion beam or transmission electron microscopy). It is also possible to create a hole in a thin Teflon substrate across which you span an artificial membrane. Within this membrane you can add alpha-hemolysin. This complex sits in the membrane and effectively makes the 1.5 nm wide nanopore. Because in our lab we aim to measure DNA-protein complexes, which are larger than the diameter of this pore, we are looking into other existing biological nanopores

Project description

This assignment focuses on testing other membrane complexes (MscL, aquaporins, …) following a protocol very similar to that used for alpha-hemolysin. You will use a special device for creating artificial membranes in which the membrane complexes can be inserted. Essential for the application targeted for, is the stability of the protein. This is whether the protein complex remains open for a longer time. If that turns out to be successful the next step is to add DNA-protein complexes to test whether these can be detected.

Literature