Poster abstracts

Poster number 27 submitted by Cho-Ying Chuang

Multi-color high-resolution fleezers development and measurement of RNA polymerase transcription

Cho-Ying Chuang (Department of Physics and Astronomy, Michigan State University, East Lansing, MI 48864, USA), Matthew Zammit (Department of Physics and Astronomy, Michigan State University, East Lansing, MI 48864, USA), Abhishek Mazumder (Waksman Institute and Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ 08854, USA), Richard H. Ebright (Waksman Institute and Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ 08854, USA), Matthew J. Comstock (Department of Physics and Astronomy, Michigan State University, East Lansing, MI 48864, USA)

Abstract:
Here we present developments in multi-color high-resolution fleezers instrumentation and methods. We have developed a high-resolution fleezers instrument with three-color confocal fluorescence microscopy. To reduce photobleaching due to traps, timeshared dual optical traps were interlaced and synchronized with three fiber coupled fluorescence excitation lasers (473 nm, 532 nm, and 633 nm) and three single-photon counting fluorescence detectors. We show a number of new fluorescence detection schemes useful both for measurement and instrument alignment including: We have developed a fleezers compatible method to image inorganic fluorescent quantum dots. Dramatically reduced photobleaching compared to organic fluorophores allows fleezers imaging and precise fluorophore localization. To remove the fluorescence background from beads, we combined a multi-channel flow chamber and internal hairpin binding construct to perform 'cryptic binding' experiments. We have measured fluorophore photobleaching lifetimes with a set of fluorophores for a variety of excitation powers in a range of buffer conditions. Finally, we present preliminary RNA polymerase transcription and fluorescence measurements. We show that the high-resolution fleezers instrument accurately measures transcription for greater than 1 kb with no difficulties. We discuss fluorescence challenges.

References:
Comstock MJ, et al. (2015) Direct observation of structure-function relationship in a nucleic acid-processing enzyme. Science 348(6232):352–354

Keywords: RNA polymerase, single molecule fluorescence, optical trapping