| Abstract [eng] |
Single-molecule techniques, which are designed for protein-DNA interaction studies, allow to gather valuable information about every individual molecule that is present in the sample. Employment of classical biochemical methods, which are based on ensemble measurements, prevents the obtainment of this useful information that is typically covered under the overall average of contribution coming from all molecules. However, vast majority of such single-molecule techniques faces with the problem of low throughput because of their incapability to manipulate more than one investigatory biomolecule at a time. Due to its principle of effective functionality “DNA Curtains” technology, which is meant for protein and DNA interaction studies at a single molecule level, effectively treats the aforementioned problem and grants a possibility to collect a large amount of statistical data during a single experiment. Recently, we have developed a platform of soft “DNA Curtains” that is based on the alternative strategy of immobilization of DNA molecules on the surface. In comparison with the other variants of traditional “DNA Curtains” tool, our experimental platform not only retains high throughput, the main attribute of this technology, but it also has some other important advantages such as low cost, simplicity and technological accessibility. Nevertheless, an efficient practical applicability of our soft “DNA Curtains” is limited by three major drawbacks – insufficient optimization of factors that influence the quality of surface-printed protein lines the most, relatively short duration of immobilization of surface-fixed individual DNA molecules and undefined orientation of biotinylated DNA molecules that are double tethered on the surface. In this work, we improved our technology of soft “DNA Curtains”. By optimizing the printing pressure, which acts during the stage of protein lift-off microcontact printing, employing traptavidin for nanopatterning of chemically modified glass surface and functionalizing the ends of DNA molecules with labels of distinct specifity – biotin and digoxigenin, respectively – we developed longer duration of immobilization of surface-fixed DNA molecules-predetermining and defined orientation of immobilized DNA molecules-ensuring version of our platform – oriented soft “DNA Curtains”. Ultimately, by employing Cas9 endonuclease, we demonstrated that this experimental tool can be successfully implemented in single protein-DNA interaction studies. |
