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Solid state nanopores

Solid state nanopores are a new type of nanoscale device inspired by protein channels that can be utilized as DNA sensors. They present a powerful tool for the investigation of individual biopolymers. Compared to their biological counterparts, synthetic nanopores offer the advantages of size control, increased stability, and the potential of device integration.

In a typical configuration, a membrane containing a single nanopore separates two compartments of saline solutions. When a voltage difference is applied between these chambers, electrically charged molecules flow through the nanopore and an electric current is measured. Individual DNA molecules, which are a negatively charged, are pulled by the electric field through the nanopore and a change in the current is recorded.

 

a) A DNA molecule is driven through a nanopore by the effect of an electric field. b) As the DNA molecule crosses the nanopore, the current decreases (experiment in a 10 nm nanopore with l-DNA in 0.5 M KCl)

We enhance the nanopore performance and capabilities by combining the use of nanopores with laser tweezers. This experimental strategy allows for the simultaneous measurement of ionic current, DNA position, and external forces that drive the translocation process. The capture of DNA inside the nanopore can be detected as changes in both the ionic conductance and the force. This technique allows for the first time the measurement of the binding and polymerization forces of a single nucleoprotein filament. We bind RecA (a multifunctional protein that plays an essential role in the process of recombinational DNA repair) to a single- or double-stranded DNA molecule and follow its polymerization against a constraining force in real time. Nanopore force spectroscopy bears the major advantage that proteins need not be chemically modified in any way, in contrast to all fluorescent techniques. The unlabeled approach uses the native proteins avoiding any potential artifact. This technique allows the direct measurement of RecA polymerization forces and that of cooperative binding proteins in general, directly quantifying the interaction between proteins and DNA.

 

A DNA molecule is captured in the nanopore and RecA proteins are allowed to bind to the DNA on the trans side of the pore. A filament grows until it hits the nanopore and cannot cross it because the pore is smaller than the RecA filament. The RecA filament keeps growing, driven by thermal motion until the restoring optical force stalls the filament growth.