Poster abstracts

Poster number 91 submitted by Faqing Yuan

Fluorophore suitability for single molecule FRET and time-resolved fluorescence study on pRNA structure concerning nucleotide distance constraints

Faqing Yuan (UC BME), Hui Zhang, Dan Shu, Peixuan Guo (UC BME)

Abstract:
The bacteriophage phi29 DNA-packaging motor is the most powerful bio-motor constructed to date. The RNA component, called packaging RNA or pRNA, plays important roles in the DNA packaging process. Six pRNA subunits bind to the distal region of the connector of the procapsid and form a hexamer ring. We are interested in elucidating: 1) interactions between pRNA and other motor components, such as connector and the ATPase gp16; and 2) pRNA motion during the DNA packaging process. To this aim, single-molecule (sm-) and time-resolved (tr-) FRET (fluorescence renascence energy transfer) are conducted to calculate the distances constraints. Sm-FRET is based on the fluorescence intensity emitted by a pair of fluorophores depending on the distance between the donor and the receptor at single molecule level. While tr-FRET depends on the ensemble data instead of single molecule, it can report on photophysical events that are difficult or impossible to observe by fluorescence intensity imaging and has the ability to deconvolute multiple components and get more accurate energy transfer data. Results from these methods are complimentary. Our results indicate that the properties of fluorescent dyes need to be carefully examined so that they can be better suited for the specific experiments. Cyanine dyes, such as Cy3 and Cy5, are good sm-FRET probes for their photo-stability. Stable fluorescence curve with less fluctuation and blinking were obtained. However, the Cy3/Cy5 pair displayed complicated lifetime profiles. Alexa dyes, such as 546 and 647, displayed simple lifetime profiles; however, required different environment to achieve photo-stability and in most cases displayed fluctuated intensity curves in sm-FRET measurement. Though small variation were found, distance constraint data strongly support the global structure of the three D models of phi29 pRNA constructed in this lab (Hoeprich S, Guo P. Computer modeling of three-dimensional structure of DNA-packaging RNA (pRNA) monomer, dimer, and hexamer of Phi29 DNA packaging motor. J Biol Chem. 2002. 277(23):20794-803).

References:
Hoeprich S, Guo P. Computer modeling of three-dimensional structure of DNA-packaging RNA (pRNA) monomer, dimer, and hexamer of Phi29 DNA packaging motor. J Biol Chem. 2002. 277(23):20794-803

Keywords: phi 29, FRET, photostability