DNA Manipulation and Single-Molecule Imaging

Abstract

DNA replication, repair, and recombination in the cell play a significant role in the regulation of the inheritance, maintenance, and transfer of genetic information. To elucidate the biomolecular mechanism in the cell, some molecular models of DNA replication, repair, and recombination have been proposed. These biological studies have been conducted using bulk assays, such as gel electrophoresis. Because in bulk assays, several millions of biomolecules are subjected to analysis, the results of the biological analysis only reveal the average behavior of a large number of biomolecules. Therefore, revealing the elementary biological processes of a protein acting on DNA (e.g., the binding of protein to DNA, DNA synthesis, the pause of DNA synthesis, and the release of protein from DNA) is difficult. Single-molecule imaging allows the analysis of the dynamic behaviors of individual biomolecules that are hidden during bulk experiments. Thus, the methods for single-molecule imaging have provided new insights into almost all of the aspects of the elementary processes of DNA replication, repair, and recombination. However, in an aqueous solution, DNA molecules are in a randomly coiled state. Thus, the manipulation of the physical form of the single DNA molecules is important. In this review, we provide an overview of the unique studies on DNA manipulation and single-molecule imaging to analyze the dynamic interaction between DNA and protein.

DNA Flow-Stretch Assays for Studies of Protein-DNA Interactions at the Single-Molecule Level

“…FRET is a distance-dependent non-radiative energy transfer from the donor fluorophore to the acceptor through the dipole-dipole interaction [32]. smFRET experiments usually rely on the excitation and detection of a pair of donor and acceptor fluorophores located on the same individual molecule or two individual interacting molecules (Figure 1F) [33].…”

“…The vast majority of previously described SM biophysical techniques that can be applied for exploring protein and DNA interactions allow one to manipulate only one investigatory biomolecule at a time. The experimental design of DNA flow-stretch assays enables the observation of tens of individual fluid flow-extended DNA molecules in the same field of view of the fluorescence microscope [32]. In comparison to the others, the increased throughput of this method makes it feasible to conduct more efficient SM-level studies of dynamic protein-DNA interactions.…”