Nynke Dekker

Abstract:

Low-frequency ionic current noise in solid-state nanopores imposes a limitation on the time resolution achieved in translocation experiments. Recently, this 1/f noise was described as obeying Hooge's phenomenological relation, where the noise scales inversely with the number of charge carriers present. Here, we consider an alternative model in which the low-frequency noise originates from surface charge fluctuations. We compare the models and show that Hooge's relation gives the best description for the low-frequency noise in solid-state nanopores over the entire salt regime from 10(-3) to 1.6 M KCl.

Abstract:

The translocation of small molecules and polymers is an integral process for the functioning of living cells. Many of the basic physical, chemical, and biological interactions have not yet been studied because they are not directly experimentally accessible. We have shown that a combination of optical tweezers, single solid-state nanopores, and electrophysiological ionic current detection enable deeper insight into the behavior of polymers in confinement. Here we describe the experimental procedures that are necessary to manipulate single biopolymers in a single nanopore, not only by electrical fields, but also through mechanical forces using optical tweezers.

Abstract:

The past few years have seen the application of single-molecule force spectroscopy techniques to the study of topoisomerases. Magnetic tweezers are particularly suited to the study of topoisomerases due to their unique ability to exert precise and straightforward control of the supercoiled state of DNA. Here, we illustrate in a stepwise fashion how the dynamic properties of type IB topoisomerases can be monitored using this technique.

Abstract:

RNA-dependent RNA polymerases (RdRP) form an important class of enzymes that is responsible for genome replication and transcription in RNA viruses and involved in the regulation of RNA interference in plants and fungi. The RdRP kinetics have been extensively studied, but pausing, an important regulatory mechanism for RNA polymerases that has also been implicated in RNA recombination, has not been considered. Here, we report that RdRP experience a dramatic, long-lived decrease in its elongation rate when it is reinitiated following stalling. The rate decrease has an intriguingly weak temperature dependence, is independent of both the nucleotide concentration during stalling and the length of the RNA transcribed prior to stalling; however it is sensitive to RNA structure. This allows us to delineate the potential factors underlying this irreversible conversion of the elongation complex to a less active mode.

Abstract:

We report the successful functionalization of optically accessible nanostructures, suitable for single-molecule experiments at physiological substrate concentrations, with polyethylene glycol. Characterization of the coating in terms of roughness, protein repellence, and specific immobilization of DNA is described. We present an application of this technique in the detection of polymerase activity within nanostructures, which demonstrates the opportunities made possible through the integration of nanofabricated structures with surface functionalization.