Talk on Friday 02:15-02:30pm submitted by Mario Vieweger
Construction and Characterization of G0-G4 RNA Dendrimers with Phi29 3WJ Modules
Mario Vieweger (College of Pharmacy; College of Medicine/Department of Physiology & Cell Biology/Dorothy M. Davis Heart and Lung Research Institute, The Ohio State University, Columbus, Ohio, USA.), Ashwani Sharma (Nanobiotechnology Center; Markey Cancer Center; 3Department of Pharmaceutical Sciences; University of Kentucky, Lexington, KY 40536, USA.), Farzin Haque (College of Pharmacy; College of Medicine/Department of Physiology & Cell Biology/Dorothy M. Davis Heart and Lung Research Institute, The Ohio State University, Columbus, Ohio, USA.), Peixuan Guo (College of Pharmacy; College of Medicine/Department of Physiology & Cell Biology/Dorothy M. Davis Heart and Lung Research Institute, The Ohio State University, Columbus, Ohio, USA.)
We report the design and self-assembly of five generations (G0-G4) of monodisperse, globular, and nanoscale-sized RNA dendrimers using the ultrastable three-way junction (3WJ) motif of bacteriophage phi29 as building block. This series of dendrimer generations is assembled around a central, square-shaped RNA core with a 3WJ motif at each corner (G0). Incorporation of overhanging sticky-ends allows radial growth in a controlled and step-wise manner by addition of 3WJ layers up to G4. All five generations can be efficiently self-assembled using one-pot synthesis into monodisperse samples, which is a significant advantage over conventional dendrimers that typically require step-wise synthesis or produce heterogeneous particle sizes. Products can further be purified using Sucrose Gradient Ultracentrifugation to yield highly purified and homogeneous samples, as demonstrated by gel electrophoresis and AFM imaging. Information from structure prediction, AFM and DLS suggests a compact globular shape in solution with diameters ranging from 2.8 nm (G0) to 26 nm (G4). Thermal gradient fluorescence and gel electrophoresis (TGGE) reveal a bi-phasic assembly/disassembly mechanism in which the outer most layers form around a preformed thermodynamically stable core. This feature allows thermodynamic control over release and degradation of the external 3WJ strands that can be modified to contain targeting and therapeutic moieties. Upon incorporation of folate on the peripheral branches, RNA dendrimers showed high binding and internalization into cancer cells. RNA dendrimers are envisioned to have a major impact in targeting, disease therapy, molecular diagnostics and bioelectronics in the near future.
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Keywords: RNA Nanotechnology, dendrimers, Phi29 3WJ