University of Cincinnati, Cincinnati, Ohio 45267, USA

 

The ingenious design of the bacterial virus phi29 DNA packaging motor with an elegant and elaborate channel has inspired its application for single molecule detection and nanomedicine applications. The hub of the nanomotor is a twelve protein subunits that form a ring with a central 3.6-nm channel acting as a path for dsDNA to enter during packaging and to exit during infection. The connector was inserted into a lipid bilayer and the resulting system exhibited robust properties and generate extremely reliable, precise and sensitive conductance signatures when ions or DNA/RNA pass through the channel. Information about the structure, length and conformational dynamics can then be deduced by their characteristic dwell times during translocation and by their relative percentage in current blockades. We further demonstrate that motor exercises a one-way traffic property for dsDNA translocation from the narrower to the wider end with a valve mechanism in DNA-packaging, as demonstrated by voltage ramping, electrode polarity switching, and sedimentation force assessment. The observed single direction dsDNA transportation provide a novel system with a natural valve to control dsDNA loading and gene delivery in bioreactors, liposomes, or high throughput DNA sequencing apparatus.

         Explicit engineering of the phi29 connector is possible due to its available crystal structure. Accordingly, we generated cysteine mutations within the inner wall of the connector channel and conjugated chemicals to the cysteine residues in real time. The sequential interaction of model analytes to each cysteine induced step-wise blocks (a single molecule per block) with defined current generate a unique fingerprint. The results demonstrate the feasibility of generating a new class of receptors for detecting biomolecules or chemicals at extremely low concentrations in the presence of many contaminants. This robust membrane-embedded phi29 connector system will have a wide range of applications in biotechnology, disease diagnosis, pathogen detection, homeland security, drug/toxin screening, and environmental sensing of chemical contaminations.

Ref: 1.Wendell, D., Jing, P., Geng, J., Subramaniam, V., Lee, T. J., Montemagno, C. & Guo P. 2009. Translocation of double stranded DNA through membrane adapted phi29 motor protein nanopore. Nature Nanotechnology. 4:765. [link][pdf]

2. Jing P, Haque F, Shu D, Montemagno C. & Guo P. 2010. One-Way Traffic of a Viral Motor Channel for Double-Stranded DNA Translocation. Nano Lett, 2010, 10, 3620.[link] [pdf]

3. Guo P. 2010.The emerging field of RNA nanotechnology. Nature Nanotechnology. 5, 833 [link] [pdf] 

4. Shu D, Shu Y, Haque F, Abdelmawla S and Guo, P. Thermodynamically stable RNA three-way junction as a platform for constructing multifunctional nanoparticles for delivery of therapeutics. Nature Nanotechnology. in press.

       Dr. Peixuan Guo is the Dane & Mary Louise Miller Endowed Chair of Biomedical Engineering, and Director of the NIH Nanomedicine Development Center located at the University of Cincinnati

         He received his Ph.D. in Microbiology with training in biophysics from the University of Minnesota in 1987; as a postdoct at NIH before joining Purdue University as an assistant professor in 1990.  tenured in 1993,  full Professor in 1997, and was honored as a Purdue Faculty Scholar in 1998. He was recruited to University of Cincinnati as endowed chair in 2007. 

         He constructed phi29 DNA packaging motor (PNAS, 1986), discovered phi29 motor pRNA (Science, 1987), assembled infectious dsDNA viruses (J Virology, 1995), discovered pRNA hexamer (Mol Cell, 1998), pioneered RNA nanotechnology (Mol Cell, 1998, JNN, 2003; Nano Lett., 2004,2005; Nature Nanotechnology,2010). His lab built a dual imaging system to detect single-fluorophores (EMBO J, 2007; RNA, 2007), incorporated the phi29 motor channel into a lipid membrane (Nature Nanotechnology, 2009) for single molecule sensing with potentials for high throughput dsDNA sequencing.

         He received the Pfizer Distinguished Faculty Award in 1995; the Purdue Faculty Scholar award in 1998; the Purdue Seed Award in 2004, 2005, and 2007; the Lions Club Cancer Research Award in 2006; and COV Distinguished Alumni of the University of Minnesota in 2009. He is an editor or board member of five nanotech journals. His work has been reported hundreds of times over the radio or TV such as ABC and NBC, and featured in Newsletters or websites of NIH, NSF, MSNBC, NCI and ScienceNow etc. He was a member of two prominent national nanotech initiatives sponsored by NIST, NIH, NSF and National Council of Nanotechnology; panelist of DOD medical assessment workshop; director of one NIH Nanomedicine Development Center from 2006 to 2011, member of the NIH NDC Steering Committee from 2006-2010, member of the review panel (site-visit) of NCI Intramural Research Program in 2010, and member of the Alliance Coordination and Governance Committee, a $150 Million program of Nanotechnology in Cancer.