2020/06/17(Wed) 10:30 -11:30 五樓第一會議室 5F, 1st Meeting Room
Title
Multicolor fluorescence optical tweezers methods and applications to nucleic acid folding
Speaker
Dr. Cho-Ying Chuang (Michigan State University)Abstract
Nucleic acids and proteins are fundamental molecular units of life. To understand their properties, we need powerful tools that allow investigation at the single-molecule level. Over the past three decades, the
development of single-molecule force and fluorescence techniques have provided us new knowledge that was previously unattainable through ensemble measurements. However, we lack methods that allow us to
precisely measure the mechanical properties of these molecules while visually detecting multiple molecules at the same time.
In this talk, we present instrumentation advances that combine angstrom-resolution optical tweezers and multicolor confocal fluorescence spectroscopy along with automated single molecule assembly. We maximize the information obtained in single-molecule measurements by pushing the techniques to more precise and more complex. Taking advantage of these methods and instrumentation advances, we
investigate the folding and unfolding of a DNA secondary structure: thrombin-binding aptamer (TBA) G-quadruplex. Studying the kinetics of G-quadruplex formation is essential for understanding telomere
regulation (the ends of chromosome) and therapeutic approaches for diseases. TBA is the smallest Gquadruplex. Although many experiments and simulations have been done on G-quadruplex, the small
size and low stability make it very difficult to observe folding and unfolding of TBA directly. Our highresolution optical tweezers have the sensitivity and stability to directly observe TBA at very low forces.
We found that with increasing force, the folding rate decreased and the unfolding rate increased. Our work demonstrates that at a given force, the TBA formation is facilitated by metal ions and is stabilized by thrombin. It also indicates that the equilibrium force increased as KCl concentration increased. From a detailed analysis of the folding and unfolding rate constants vs applied force, we were able to detect a single transition state conserved across all conditions and identify the structure of the transition state as the G-triplex structure.
Language
演講語言 (Language): in English