| Popis: |
Magnetic and optical tweezers have grown into a key tool when examining single molecules of DNA. However, it has not so far been possible to directly manipulate DNA through an applied force or torque while imaging its full contour length. In this work, a novel combined magnetic and optical tweezing tool developed at the University of York [1] has been combined with superresolution fluorescence microscopy [2] and a novel experimental geometry in which the DNA is held horizontally to the field of view. One end of the DNA is attached to an anchor bead on the surface of the coverslip through multiple digoxigenin-antidigoxigenin interactions, while the other is attached to a smaller superparamagnetic bead with multiple biotin-streptavidin bonds. The smaller bead may then be trapped with a near-infrared laser trap, rotated with a magnetic field, or both. Back focal plane detection using the IR laser is employed to quantify trap stiffness and rotational frequency, as well as applied force. The DNA is fluorescently imaged with the intercalating dye YOYO-1, which may be localised to within 30-40nm. Experimental data can be compared with coarse-grained and atomistic simulations to give detailed information about the molecule. Application of this methodology will allow real-time imaging and quantification of key DNA processes such as plectoneme formation and DNA overstretching, and the apparatus may also be used as a tool to monitor molecular machines with a view toward focussed, bottom-up drug design.[1]. Zhou, Z., Miller, H., Wollman, A., & Leake, M. (2015). Developing a New Biophysical Tool to Combine Magneto-Optical Tweezers with Super-Resolution Fluorescence Microscopy. Photonics, 2(3), 758-772.[2] Miller, H., Zhou, Z., Wollman, A. J. M., & Leake, M. C. (2015). Superresolution imaging of single DNA molecules using stochastic photoblinking of minor groove and intercalating dyes. Methods, 88, 81-88. |
