Integrating Optical Tweezers, DNA Tightropes, and Single-Molecule Fluorescence Imaging: Pitfalls and Traps

• Published in: Methods in enzymology Vol. 582; p. 171
• Main Authors: Wang, J, Barnett, J T, Pollard, M R, Kad, N M
• Format: Journal Article
• Language: English
• Published: United States 2017
• Subjects: DNA Repair - genetics; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - isolation & purification; Microscopy, Fluorescence; Nanotechnology; Optical Imaging - methods; Optical Tweezers; Quantum Dots - chemistry; Single Molecule Imaging - methods; Nucleotide excision repair; Laser tweezers; Fluorescence imaging; Quantum dots; DNA repair; Single molecule
• ISSN: 1557-7988
• DOI: 10.1016/bs.mie.2016.08.003

Fluorescence imaging is one of the cornerstone techniques for understanding how single molecules search for their targets on DNA. By tagging individual proteins, it is possible to track their position with high accuracy. However, to understand how proteins search for targets, it is necessary to elongate the DNA to avoid protein localization ambiguities. Such structures known as "DNA tightropes" are tremendously powerful for imaging target location; however, they lack information about how force and load affect protein behavior. The use of optically trapped microstructures offers the means to apply and measure force effects. Here we describe a system that we recently developed to enable individual proteins to be directly manipulated on DNA tightropes. Proteins bound to DNA can be conjugated with Qdot fluorophores for visualization and also directly manipulated by an optically trapped, manufactured microstructure. Together this offers a new approach to understanding the physical environment of molecules, and the combination with DNA tightropes presents opportunities to study complex biological phenomena.