Biomedical subjects

N H Dekker

Publications and source records attributed to N H Dekker.

5 recordsLinked to original sources

PubMed2006

Nanobubbles in solid-state nanopores.

From conductance and noise studies, we infer that nanometer-sized gaseous bubbles (nanobubbles) are the dominant noise source in solid-state nanopores. We study the ionic conductance through solid-state nanopores as they are moved through the focus of an infrared laser beam. The resulting conductance profiles show strong variations in both the magnitude of the conductance and in the low-frequency noise when a single nanopore is measured multiple times. Differences up to 5 orders of magnitude are found in the current power spectral density. In addition, we measure an unexpected double-peak ionic conductance profile. A simple model of a cylindrical nanopore that contains a nanobubble explains the measured profile and accounts for the observed variations in the magnitude of the conductance.

Electric Conductivity↗

PubMed2005

Single-molecule measurements of the persistence length of double-stranded RNA.

Over the past few years, it has become increasingly apparent that double-stranded RNA (dsRNA) plays a far greater role in the life cycle of a cell than previously expected. Numerous proteins, including helicases, polymerases, and nucleases interact specifically with the double helix of dsRNA. To understand the detailed nature of these dsRNA-protein interactions, the (bio)chemical, electrostatic, and mechanical properties of dsRNA need to be fully characterized. We present measurements of the persistence length of dsRNA using two different single-molecule techniques: magnetic tweezers and atomic force microscopy. We deduce a mean persistence length for long dsRNA molecules of 63.8 +/- 0.7 nm from force-extension measurements with the magnetic tweezers. We present atomic force microscopy images of dsRNA and demonstrate a new method for analyzing these, which yields an independent, yet consistent value of 62 +/- 2 nm for the persistence length. The introduction of these single-molecule techniques for dsRNA analysis opens the way for real-time, quantitative analysis of dsRNA-protein interactions.

Biophysics↗

PubMed2004

Joining of long double-stranded RNA molecules through controlled overhangs.

We describe two methods for creating long (>1 kb) dsRNA molecules with specific, user-controlled overhangs for efficient hybridization and ligation. The two methods create double-stranded RNA (dsRNA) molecules with 5' overhangs or with 3' overhangs using T7 RNA polymerase (T7 RNAP) in transcription reactions of carefully designed PCR products. Primers utilized in the PCR reactions provide the template for the desired dsRNA overhangs. These methods provide complete control of the length and the sequence of the overhangs. This supplies a tool which is particularly lacking in dsRNA biochemistry given the absence of restriction endonucleases active on these substrates.

DNA↗

PubMed2003

Thermophilic topoisomerase I on a single DNA molecule.

Control of DNA topology is critical in thermophilic organisms in which heightened ambient temperatures threaten the stability of the double helix. An important role in this control is played by topoisomerase I, a member of the type IA family of topoisomerases. We investigated the binding and activity of this topoisomerase from the hyperthermophilic bacterium Thermotoga maritima on duplex DNA using single molecule techniques, presenting it with various substrates such as (+) plectonemes, (-) plectonemes, and denaturation bubbles. We found the topoisomerase inactive on both types of plectonemes, but active on denaturation bubbles produced at increased stretching forces in underwound DNA. The relaxation rate depended sensitively on the applied force and the protein concentration. These observations could be understood in terms of a preference of the topoisomerase for single-stranded DNA over double-stranded DNA and allowed for a better understanding of activity of the topoisomerase in bulk experiments on circular plasmids. Binding experiments on a single duplex molecule using a mutant unable to perform cleavage confirmed this interpretation and suggested that T.maritima topoisomerase I behaves like an SSB by lowering the denaturation threshold of underwound DNA. Finally, experiments with a unique single-stranded DNA showed that both ends of the cleaved DNA are tightly maintained by the enzyme, supporting an enzyme-bridged mechanism for this topoisomerase.

DNA Topoisomerases, Type I↗

PubMed2002

The mechanism of type IA topoisomerases.

The topology of cellular DNA is carefully controlled by enzymes called topoisomerases. By using single-molecule techniques, we monitored the activity of two type IA topoisomerases in real time under conditions in which single relaxation events were detected. The strict one-at-a-time removal of supercoils we observed establishes that these enzymes use an enzyme-bridged strand-passage mechanism that is well suited to their physiological roles and demonstrates a mechanistic unity with type II topoisomerases.

Biophysical Phenomena↗