Poster abstracts
Poster number 45 submitted by Hui Zhang
Single-molecule analysis of structure and assembly of RNA nanoparticles
Hui Zhang (College of Pharmacy, The Ohio State University), Peixuan Guo (College of Pharmacy, The Ohio State University)
Abstract:
A customized dual-channel single molecule imaging system has been constructed to study the phi29 DNA-packaging motor that is geared by a hexameric pRNA ring. The motor 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 stoichiometry and the 3D structure of the pRNA is both fundamentally and practically important. The imaging system with single fluorophore sensitivity and multicolor detection ability is the combination of a low-temperature (-80 °C) sensitive electron multiplied CCD camera with both objective- and prism-type TIRF (Total Internal Reflection Fluorescence) mechanism. A laser combiner was introduced to facilitate simultaneous multi-color imaging. Two lasers with different wavelengthes were delivery synchronically via an optic fiber to the sample chamber with TIRF capability. Single molecule photobleaching combined with binomial distribution analysis clarified the stoichiometry of the pRNA on active motors. Further crystallography and biophysics studies confirmed that the pRNA forms a hexamer ring on the motor. The ability of using single molecule FRET in distance determination within the nanomotor was tested. Single molecule FRET study was carried out to investigate the structure of the pRNA dimer. Ten pRNA monomers labeled with single donor/acceptor fluorophore pairs 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 provided the distance constraints to refine the previously reported 3D model of the pRNA dimer. The analysis of the pRNA structure will help to improve the designs of therapeutic RNA nanoparticles using pRNA as a platform.
References:
1) Zhang H, Guo P. 2014. Single molecule photobleaching (SMPB) technology for counting of RNA, DNA, protein and other molecules in nanoparticles and biological complexes by TIRF instrumentation. Methods. 67(2):169-76.
2) Zhang H, Endrizzi J, Shu Y, Haque F, Sauter C, Shlyakhtenko L, Lyubchenko Y, Guo P, Chi Y. 2013. Crystal structure of 3WJ core revealing divalent ion-promoted thermostability and assembly of the Phi29 hexameric motor pRNA. RNA. 19(9):1226-37.
3) Shu D, Zhang H, Petrenko R, Meller J, and Guo P. 2010. Dual-Channel Single-Molecule Fluorescence Resonance Energy Transfer to Establish Distance Parameters for RNA Nanoparticles. ACS Nano. 4:6843.
4) Shu D, Zhang H, Jin J, Guo P, 2007. Counting of six pRNAs of phi29 DNA-packaging motor with customized single-molecule dual-view system. The EMBO Journal. 26:527.
Keywords: Single molecule, TIRF imaging, RNA structure