Probing concentration-dependent behavior of DNA-binding proteins on a single-molecule level illustrated by Rad51.

Low throughput is an inherent problem associated with most single-molecule biophysical techniques. We have developed a versatile tool for high-throughput analysis of DNA and DNA-binding molecules by combining microfluidic and dense DNA arrays. We use an easy-to-process microfluidic flow channel system in which dense DNA arrays are prepared for simultaneous imaging of large amounts of DNA molecules with single-molecule resolution. The Y-shaped microfluidic design, where the two inlet channels can be controlled separately and precisely, enables the creation of a concentration gradient across the microfluidic channel as well as rapid and repeated addition and removal of substances from the measurement region. A DNA array stained with the fluorescent DNA-binding dye YOYO-1 in a gradient manner illustrates the method and serves as a proof of concept. We have applied the method to studies of the repair protein Rad51 and could directly probe the concentration-dependent DNA-binding behavior of human Rad51 (HsRad51). In the low-concentration regime used (100 nM HsRad51 and below), we detected binding to double-stranded DNA (dsDNA) without positive cooperativity.

A study of the enhanced sensitizing capacity of a contact allergen in lipid vesicle formulations.

The growing focus on nanotechnology and the increased use of nano-sized structures, e.g. vesicles, in topical formulations has led to safety concerns. We have investigated the sensitizing capacity and penetration properties of a fluorescent model compound, rhodamine B isothiocyanate (RBITC), when administered in micro- and nano-scale vesicle formulations. The sensitizing capacity of RBITC was studied using the murine local lymph node assay (LLNA) and the skin penetration properties were compared using diffusion cells in combination with two-photon microscopy (TPM). The lymph node cell proliferation, an indicator of a compounds sensitizing capacity, increased when RBITC was applied in lipid vesicles as compared to an ethanol:water (Et:W) solution. Micro-scale vesicles showed a slightly higher cell proliferative response compared to nano-scale vesicles. TPM imaging revealed that the vesicle formulations improved the skin penetration of RBITC compared to the Et:W solution. A strong fluorescent region in the stratum corneum and upper epidermis implies elevated association of RBITC to these skin layers when formulated in lipid vesicles. In conclusion, the results indicate that there could be an elevated risk of sensitization when haptens are delivered in vehicles containing lipid vesicles. Although the size of the vesicles seems to be of minor importance, further studies are needed before a more generalized conclusion can be drawn. It is likely that the enhanced sensitizing capacity is a consequence of the improved penetration and increased formation of hapten-protein complexes in epidermis when RBITC is delivered in ethosomal formulations.

Incorporation of a transmembrane protein into a supported 3D-matrix of liposomes for SPR studies.

Surface analytical tools as surface plasmon resonance (SPR) have become increasingly important in biomedical research since they offer high detection sensitivity compared to traditional biomedical methods. For the use of SPR as a biomedical research tool there is a need to immobilize the reactants to a solid sensor surface. It is nowadays fairly straightforward to immobilize various reactants and hydrophilic proteins to a solid sensor surface and SPR has successfully been used in several applications using such proteins when studying various protein interactions. When using SPR for the analysis of transmembrane proteins the immobilization onto the solid surface becomes more difficult. Transmembrane proteins are more sensitive to the surroundings and need to be incorporated into a structure where it can reside in a natural environment. Supported liposomes offer such environment. In this chapter a new method is presented where multilayers of such supported liposomes are used to immobilize transmembrane proteins onto a solid sensor surface which is suitable for use in SPR detection.

A microfluidic device for reversible environmental changes around single cells using optical tweezers for cell selection and positioning.

Cells naturally exist in a dynamic chemical environment, and therefore it is necessary to study cell behaviour under dynamic stimulation conditions in order to understand the signalling transduction pathways regulating the cellular response. However, until recently, experiments looking at the cellular response to chemical stimuli have mainly been performed by adding a stress substance to a population of cells and thus only varying the magnitude of the stress. In this paper we demonstrate an experimental method enabling acquisition of data on the behaviour of single cells upon reversible environmental perturbations, where microfluidics is combined with optical tweezers and fluorescence microscopy. The cells are individually selected and positioned in the measurement region on the bottom surface of the microfluidic device using optical tweezers. The optical tweezers thus enable precise control of the cell density as well as the total number of cells within the measurement region. Consequently, the number of cells in each experiment can be optimized while clusters of cells, that render subsequent image analysis more difficult, can be avoided. The microfluidic device is modelled and demonstrated to enable reliable changes between two different media in less than 2 s. The experimental method is tested by following the cycling of GFP-tagged proteins (Mig1 and Msn2, respectively) between the cytosol and the nucleus in Saccharomyces cerevisiae upon changes in glucose availability.

Characterization of a proton pumping transmembrane protein incorporated into a supported three-dimensional matrix of proteoliposomes.

Surface analytical tools have gained interest in the bioanalytical field during recent years because they offer the possibility of more detailed investigations of biomolecular interactions. To be able to use such tools, the biomolecules of interest must be immobilized to a surface in a functioning way. For small water-soluble biomolecules, the surface immobilization is quite straightforward, but it has been shown to be difficult for large transmembrane proteins. In those cases, the solid surface often has a negative influence on the function of the transmembrane proteins. In this article, we present a new approach for surface immobilization of transmembrane proteins where the proteins were immobilized on a surface in a proteoliposome multilayer structure. The surface-binding events and the structure of the surface-immobilized proteoliposomes were monitored using both the quartz crystal microbalance with dissipation monitoring (QCM-D) and surface plasmon resonance (SPR) techniques. With this multilayer proteoliposome structure, it was possible to detect trypsin digestion of the transmembrane protein proton translocating nicotinamide nucleotide transhydrogenase in real time using SPR. The results from the combined SPR and QCM-D analysis were confirmed by fluorescence microscopy imaging of the multilayer structure and activity measurements of transhydrogenase. These results showed that the activity of transhydrogenase was significantly decreased in the bottom layer, but in the subsequent proteoliposome layers 90% of the activity was retained compared with bulk measurements. These results emphasize the importance of an immobilization strategy where the transmembrane proteins are lifted off the solid surface at the same time as the amount of protein is increased. We consider this new method for surface immobilization of transmembrane proteins to meet these demands and that the method will improve the possibility to use a variety of surface analytical tools for the analysis of interactions involving transmembrane proteins in the future.

Long-distance lateral diffusion of human Rad51 on double-stranded DNA.

Rad51 is the primary eukaryotic recombinase responsible for initiating DNA strand exchange during homologous recombination. Although the subject of intense study for over a decade, many molecular details of the reactions promoted by Rad51 and related recombinases remain unknown. Using total internal reflection fluorescence microscopy, we directly visualized the behavior of individual Rad51 complexes on double-stranded DNA (dsDNA) molecules suspended in an extended configuration above a lipid bilayer. Here we show that complexes of Rad51 can bind to and slide freely along the helical axis of dsDNA. Sliding is bidirectional, does not require ATP hydrolysis, and displays properties consistent with a 1D random walk driven solely by thermal diffusion. The proteins move freely on the DNA for long periods of time; however, sliding terminates and the proteins become immobile upon encountering the free end of a linear dsDNA molecule. This study provides previously uncharacterized insights into the behaviors of human Rad51, which may apply to other members of the RecA-like family of recombinases.

Organized arrays of individual DNA molecules tethered to supported lipid bilayers.

An unappreciated aspect of many single-molecule techniques is the need for an inert surface to which individual molecules can be anchored without compromising their biological integrity. Here, we present new methods for tethering large DNA molecules to the surface of a microfluidic sample chamber that has been rendered inert by the deposition of a supported lipid bilayer. These methods take advantage of the "bio-friendly" environment provided by zwitterionic lipids, but still allow the DNA molecules to be anchored at fixed positions on the surface. We also demonstrate a new method for constructing parallel arrays of individual DNA molecules assembled at defined positions on a bilayer-coated, fused silica surface. By using total internal reflection fluorescence microscopy to visualize the arrays, it is possible to simultaneously monitor hundreds of aligned DNA molecules within a single field-of-view. These molecular arrays will significantly increase the throughput capacity of single-molecule, fluorescence-based detection methods by allowing parallel processing of multiple individual reaction trajectories.

Utilizing adsorbed proteoliposomes trapped in a non-ruptured state on SiO2 for amplified detection of membrane proteins.

The quartz crystal microbalance with dissipation (QCM-D) technique was used to monitor the formation of supported phospholipid bilayers (SPBs) on SiO2 using proteoliposomes with reconstituted proton translocating nicotinamide nucleotide transhydrogenase (TH). Exposure of the surface to such proteoliposomes creates a lipid film composed of a mixture of proteolipid bilayers and adsorbed non-ruptured proteoliposomes, where the fraction of the latter is reduced if the TH-liposomes are pretreated with trypsin to remove the water soluble domains of TH [Langmuir 19 (2003) 842]. In the present work, the latter study is complemented by investigating the influence of trypsin treatment of the mixed adlayer (proteolipid bilayer + non-ruptured proteoliposomes) after adsorption on the surface. This demonstrates how trypsin-cleavage induced rupture of adsorbed TH-liposomes can be utilized to detect the presence of less than 0.04 pmol/cm2 of immobilized TH.