Poster abstracts

Poster number 84 submitted by Hui Zhang

Dual-channel single-molecule imaging of pRNA on phi29 DNA-packaging motor

Hui Zhang (Department of Biomedical Engineering, University of Cincinnati), Dan Shu (Department of Biomedical Engineering, University of Cincinnati), Roman Petrenko (Department of Physics, University of Cincinnati), Taejin Lee, Feng Xiao, Jarek Meller, Peixuan Guo (Department of Biomedical Engineering, University of Cincinnati)

Abstract:
Bacteriophage phi29 DNA-packaging motor is geared by six packaging RNAs (pRNA). The pRNA molecules have been reported to serve as building blocks in RNA nanotechnology, and as vehicles for specific delivery of therapeutics to treat cancers and viral infections. The understanding of the 3D structure of pRNA and its location and positioning on the motor are both fundamentally and practically important. A customized single-molecule dual-color imaging system has been constructed to study the structures of pRNA molecules. The system is the combination of a low-temperature (-80 ¡ãC) sensitive electron multiplied CCD camera and the prism-type total internal reflection mechanism. A laser combiner was introduced to facilitate simultaneous dual-channel imaging. It has been applied to study the structure, stoichiometry, distance and function of the phi29 DNA packaging motor. Single molecule photobleaching combined with binomial distribution analysis clarified the stoichiometry of pRNA on the motor and elucidated the mechanism of pRNA hexamer assembly. The feasibility of the single-molecule imaging system was demonstrated in studies of single-molecule FRET. Distance rulers made of dual-labeled dsDNA and RNA/DNA hybrids were used to evaluate the system by determining the distance between one FRET pair. The single-molecule FRET was also applied to the reconstructed the 3D structure of phi29 motor pRNA monomers and pRNA dimers. Ten pRNA monomers labeled with single donor or acceptor fluorophore at various locations were constructed, and eight partner pairs were assembled into dimers. FRET signals were detected for six dimers and utilized to assess the distance between each donor/acceptor pair. The results provide the distance constraints for 3D computer modeling of phi29 DNA packaging motor. We have also re-engineered the energy conversion protein, gp16, of phi29 motor for single fluorophore labeling to facilitate the single molecule studies of motor mechanism. The potential applications of single-molecule high-resolution imaging with photobleaching (SHRImP) and single molecule high resolution with co-localization (SHREC) approaches to the study of the phi29 nanomotor were also investigated.

Keywords: Bacteriophage phi29, Dual-channel imaging, Single molecule imaging