1. DNA binding and bending sensor
The genetic information of any organism is stored in a very remarkable molecule known as DNA. Since DNA is such an essential part of every living cell, many different enzymes interact either directly or indirectly with it. The work in our lab focuses on the maintenance of DNA by specialised DNA polymerases, which fill short gaps in DNA with high accuracy. In order to study the interactions between the DNA and the polymerase, we have developed a flourescently labelled DNA sensor, which changes its FRET signal upon binding of the polymerase to gapped DNA hinting a large conformational change of the DNA involved. The work is done in collaboration with Dr. Tim Craggs and the Kapanidis lab in Oxford.
2. Increasing the throughput of single-molecule-fluorescence measurements
There are two major techniques to study fluorescently labelled molecules: Confocal microscopy in solution, in which single molecules freely diffuse through a femtoliter-sized detection volume, and TIRF microscopy, in which hundreds of surface-immobilised molecules are imaged with a sensitive camera. By using specially designed nanofluidic devices, we are planning to combine the advantages of both methods: a high time resolution for studying fast conformational changes (as is the strength of confocal microscopy) with the ability to monitor many individual molecules in parallel (as is only possible with image-based detection of fluorescence). The work is done in collaboration with Dr. Klaus Mathwig and the Lemay group at Twente University.
3. Binding and dissociation of DNA polymerases to DNA
Acceptor labelled DNA is immobilized on a PEGylated glass cover-slide. DNA polymerase I diffuses in high concentration above the surface looking for a 3’ end of a DNA primer to bind. Successful binding of the polymerase leads to an energy transfer from the donor dye attached to the polymerase to the acceptor attached to the DNA. Due to the relatively low processivity of DNAP I the polymerase dissociates after a short time. This assay allows the determination of binding and dissociation constants under different conditions.
4. Illuminating plant hormone responses at the single-molecule level
The small signalling molecule auxin plays a key role in regulating cell division, elongation and differentiation in plants. Cellular responses on auxin are mediated via proteins belonging to the AUXIN RESPONS FACTOR (ARF) family that represent DNA-binding transcription factors. Recently, the group of Dolf Weijers [link] has revealed a novel structure-based model of ARF protein action and DNA-binding specificity by showing that regulation of target genes requires both protein-DNA interaction as well as protein dimerization. Although these crystal structures show the dimerization of the ARF DNA-binding-domain (DBD) in absence and presence of DNA, dynamic information on protein dimerization as well as potential interactions of the full-length protein are still missing.
In this project we aim to develop and utilise new fluorescence-based assays to quantify inter and intra-molecular interactions in the auxin mediated signalling pathway on the single-molecule level. By applying techniques such as single-molecule Förster resonance energy transfer (smFRET) we will characterise the binding kinetics of various ARF-DBD proteins to DNA whilst monitoring structural changes of the DNA and the associated proteins. (The project is funded by the EPS graduate school).
5. Probing anisotropic food structures using single-particle diffusometry
In order to feed the world population in a more sustainable manner, many of the current food products will have to be reformulated, ideally by using plant-derived materials. As consumers are unlikely to compromise on sensorial quality (texture), new food structures need to be designed to mimic the mouthfeel of established foods. The rational redesign of these materials, however, is currently hampered by our limited knowledge on the relationship between texture and the underlying multi-scale structural architectures. Especially the role of scale-dependent structural anisotropy, a major determinant of the texture of meat and its plant-based alternatives, is only poorly understood and difficult to probe at the sub-μm length scale.
In recent years, “nanoparticle diffusometry” has been introduced as a broader technological framework in which self-diffusing nanoparticles allow inferring local solvent viscosity, local network elasticity and network heterogeneity. This characterisation is primarily achieved by analysing the time-dependency of the mean-square displacement of the probe experimentally obtained by, for example, single-particle tracking (SPT). SPT can provide both sub-micrometre spatial and sub-millisecond temporal resolution in heterogeneous samples. These unique features of SPT have not yet been exploited for quantification of anisotropic biopolymer networks in food materials. (The project is funded by the VLAG graduate school).