Poster abstracts

Poster number 11 submitted by Dana Driver

Construction of AFM Imaging of Hexameric pRNA of phi29 DNA Packaging Motor by RNA Nanotechnology

Dana Driver (Center for RNA Nanobiotechnology and Nanomedicine; College of Pharmacy, Division of Pharmaceutics and Pharmaceutical Chemistry), Yi Shu, Peixuan Guo

Abstract:
The packaging of bacteriophage phi29 DNA genome is accomplished through the use of six RNA molecules called pRNA. pRNA serves as an interface between the gp16 ATPase and the connector protein. Without pRNA, gp16 does not bind to the procapsid and packaging does not occur. The pRNA contains upper and lower loops via a hand-in-hand integration mechanism in inter-RNA interactions. The right loop is delineated with a capital letter, while the left loop is the lower case letter. pRNA A-b’ or B-a’ with a mutation in only one hand will lead to an inactive pRNA unsuitable for packaging; however, mixing of pRNA A-b’ and B-a’ in equal concentrations to form a dimer by hand-in-hand interaction will result in active pRNA. We report the use of hand-in-hand interaction of the pRNA that allows for the construction of hexamer. The pRNA functionality for DNA packaging was assayed by DNAase protection viral assembly. Up to 8 x 10^7 PFU of infectious virions per mL were assembled in vitro, with the addition of 11 proteins derived from cloned genes and nucleic acids synthesized separately. The ultimate goal of this project is to image six pRNA strands assembled into a hexamer. A RNA triangle was fused to each pRNA strands at the 3’ end. Each RNA was produced from the PCR product using unmodified RNA transcription. Bottom-up assembly was achieved by annealing the pRNA strands and running them in 4% native gel. Through application of RNA nanotechnology, we have assembled the hexameric pRNA complex with six purified RNA molecules. Sequential addition of each RNA strands was detected by gel as evidence by the presence of RNA oligomers with gradual reduction of migration rate in native gel. Formation of RNA hexamer was confirmed by AFM imaging.

Keywords: pRNA, phi29 bacteriophage, hexamer RNA