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Project 1 Mechanism of the synthetic imitating nanomotor of phi29 (Fig. 1) (continually funded by two NIH R-01 grant since 1991) Fig. 1. Structure and function of a controllable DNA-packaging motor of bacterial virus phi29. The figure shows the tertiary bottom view (C) and side view (D) of phi29 DNA packaging motor, which is embedded in a pentagonal capsid (the green pentagon in C-D). A and B illustrate sequential action of pRNA, similar to the sequential action of six cylinders of a car engine.
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(Funded by NIH Institute of Imaging and Bioengineering R-01 grant since 2004; two grants from DOD, and two contracts from Kylin Therapeutics Inc. and Wyeth Pharmaceutical. The technology has been licensed by Kylin Therapeutics Inc. exclusively to develop my pRNA nanotechnology)
Fig.2. Methods for the construction of pRNA nanoparticles by loop/loop interaction.
Fig. 3. AFM images of the constructed pRNA nanoparticles.
Fig. 4. The phi29 pRNA hexamer as a vector for the delivery of multiple therapeutics to specific cells directed by ligands.
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Project 3 Design and application of biophysical and optical instruments including single fluorophore dual-view imaging system, both prism- and objective-type total internal reflection fluorescent microscopes, laser trap (tweezer), single molecule confocal optical system (Fig. 5-8) Fig. 5. Design of the customized top-prism single molecule dual-view total internal reflection fluorescence imaging system.
Fig. 6. Dual-view imaging of single RNA counting of procapsids containing both Cy3-pRNA and Cy5-pRNA. (A) pRNA dimer single labelled with Cy3-pRNA I and Cy5-pRNA II. (B) Typical fluorescence image of procapsids bound with dual-labeled pRNA. (C) Fluorescence intensity versus time to show photobleaching steps of procapsids with the dual-labeled dimer.
Fig. 7. Single molecule FRET study. Left: Typical fluorescence image of FRET event. Right: Typical intensity time trajectory of FRET event. The FRET efficiency and distance between two fluorophores is calculated used the above equeations.
Fig. 8. Principles of Single-molecule high-resolution imaging with photobleaching for distance measurement between two fluorophores.
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Project 4 4. Environmental single molecule sensing, pathogen detection and earl disease diagnosis using single pore sensing, single channel conductance, and nanobiotechnology (Fig. 9-12) (Funded by NIH Nanomedicine Development Center). Fig. 9. A 24 × 30 nm ellipsoid nanoparticle containing 84 subunits or 7 dodecamers of the re-engineered core protein of the phi29 DNA packaging motor was constructed. EM and analytical ultracentrifugation were employed to elucidate the structure, shape, size, and mechanism of assembly. The formation of this structure was reversible. The 84 outward-oriented C-termini were conjugated with a streptavidin binding peptide which can be used for the incorporation of markers. This further extends the application of this nanoparticle to pathogen detection and disease diagnosis by signal enhancement.
Fig. 10. Lipid directed formation of single layer connector array. (A) Illustration of the experimental approaches. (B) EM image of the array. (C) Fourier transforms and (D) corresponding Fourier projection maps of the array. (E) AFM image of tetragonal arrays. (F) and (G) Cross-sections along the axes of the 2D array in (E). (H) AFM image of the tetragonal arrays other connector.
Fig. 11. Reengineered Phi29 Motor continuously incorporated into lipid bilayers. The insertion was confirmed by the discrete current jump by single channel conductance assy.
Fig. 12. The use of fingerprint of channel conductance in DNA translocation to elucidate the mechanism of single molecule sensing. (A) The model of DNA translocation through the connector pore. (B) Current blockades due to DNA translocation though the pore. (C) Translocation of folded DNA. Top: recorded events of the current signal as fingerprints of each DNA. Bottom: dsDNA structure.
In addition, my lab has also been extensively involved in the studies of chemical conjugation, chemical crosslinking, biological computation, 3D modeling of RNA and protein structure; domain boundary quantification; computation of motor force, torque and speed, quantification of energy transduction and single pore conductance (Fig 1) (previously or currently funded by NIH and NSF). |
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(Updated on 03-09-2011) |