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Phi29 DNA-Packaging Motor for Nanomedicine

 
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Executive Summary

Fundamental understanding of nature's nanomotors has not yet effectively translated to new biomedical applications: Currently, there is no nanodevice available for actively pumping drugs, DNA, RNA, and other therapeutic molecules into specifically targeted cells. The phi29 DNA-packaging nanomotor (Fig. 1, Guo et al, Science 1987) is one of the strongest biological motors known. A key challenge is to contextualize to human systems our understanding of the unique phi29 nanomotor and its components, and to manipulate this nanomachine in an artificial environment. Our center will continue our studies of the phi29 motor to characterize its physical properties, and we will also rebuild the motor for therapeutic purposes, possibly for drug delivery. We plan to re-engineer the motor to function in lipid bilayers and other polymers, continue our detailed mechanistic studies of the re-engineered motor, and develop arrays of motors for single molecule sensing of chemicals, biomarkers, diseased cells, pathogens as well as for single pore sequencing of DNA.

 Introduction
 
Figure 1 - Schematic of the phi29 RNA nanomotorNature has been using nanoscale machines since the emergence of prokaryotic organisms and provides many examples from which biomedical researchers may harness opportunities for new nanoscale devices and approaches for medical applications. By developing artificial nanomachines, we hope to be able to interact more effectively with biological entities and to influence their behavior for desired outcomes. Bacteriophage phi29, a virus that infects bacteria, uses a nanomotor to package its genome into its capsid, creates nano-sized plugs that can resist high internal pressures of the packaged genome, has nano-tweezers that can hold on to the surface of host cells, and assembles nanoscale channels and pores for transporting its genome during replication. Further, viral capsids are protein self-assemblies that display unique and outstanding mechanical properties. We and others have intensively studied the phi29 nanomotor for years, and the major components of this motor have been identified. It is an extremely well-characterized nanomachine, providing an excellent opportunity for conducting the experiments necessary to reveal the biophysical properties of the motor and manipulating these properties for medical applications.

NDC Goals

The genome of double-stranded (ds) DNA bacteriophage phi29 is packaged into a preformed protein shell during replication. This energetically unfavorable DNA translocation process is accomplished by a DNA packaging motor geared by a hexameric ring composed of six pRNAs (packaging RNA). The pRNA assembles into dimers, trimers, and hexamers via hand-in-hand interactions between two right and left interlocking loops. The unique features of the two interlocking loops makes phi29 pRNA a promising tool for bottom-up assembly via nanomachine fabrication, pathogen detection, and co-delivery of multiple therapeutic reagents with ligands for specific cell targeting. Another essential component of the phi29 motor is a dodecameric connector composed of protein gp10, which forms a channel with a cross-section area of 10 nm2 acting as a path for dsDNA translocation. Novel and ingenious design of the motor, along with the elegant and elaborate channel, has inspired its applications into nanotechnology and nanomedicine. Incorporation of this motor or connector into a biological membrane will potentiate its applications for single molecule sensing, microreactors, gene delivery, drug loading, and sequencing of dsDNA. Based on the aforementioned exciting insight, three overarching challenges were originally elaborated for this NDC: Challenge 1. Re-Engineering the Phi29 Motor for Incorporation into Lipid Bilayers; Challenge 2. Mechanistic Studies of the Re-Engineered Membrane Adapted Motor; Challenge 3. Active Nanomotor/Metal, DNA or Silicon Arrays that Enable Drug Delivery and Diagnostics.
 
NDC Accomplishments

1. We have incorporated the connector, the core motor components of phi29 DNA packaging motor into lipid membrane, and demonstration of DNA translocation through the robust connector nanopores (the channel) by single channel conductance detection (pico amp) (Nature Nanotechnology, 2009, 4(11): 695-780).

Amazingly, the conductance of each channel was extremely uniform, consistent, and stable. Single molecule detection of DNA translocation through the motor channel also revealed that the signal for each DNA translocation was very uniform, homogeneous, and tidy. Methods for the precise and accurate counting of the number of channels per membrane have also been well established.
Figure 2 - Incorporation of connector into giant liposome

Figure 3 - The insertion of the robust connector into bilayer lipid membranes (BLM) was confirmed by conductance assay.

Figure 4 - Translocation of dsDNA through robust connector channels in BLM.

Our studies began in the context of our deep experience with the molecular biology of the phi29 viral system and its extremely well-characterized DNA packaging motor. We have now begun to utilize the multidisciplinary expertise of our NDC team to move into nanomedical applications for this system in the form of motor-studded lipid vesicles that may be used for therapeutic delivery and motor array structures for high sensitivity diagnostics, drug loading and gene delivery.

2. We Have Constructed Adjustable Ellipsoid Nanoparticles Assembled from Re-engineered Connectors of the Bacteriophage Phi29 DNA Packaging Motor (ACS Nano. 2009, 3(8):2163-70)
Figure 5 - A 24 × 30 nm ellipsoid nanoparticle containing 84 subunits or 7 dodecamers of the re-engineered core protein of the bacteriophage phi29 DNA packaging motor was constructed.

3. We have fabricated Massive Sheets of Single Layer Patterned Arrays Using Lipid Directed Reengineered Phi29 Motor Dodecamer (ACS Nano. 2009;3(1):100-7)
Figure 6 - The bottom-up assembly of patterned arrays is an exciting and important area in current nanotechnology.

4. We have modified phi29 motor components and assembled a variety of artificial motors that are functional in DNA packaging. We have been able to view individual motors with single molecule imaging instrumentation and observed the motor at work in real time (Applied Physics Letter. 2008, 93(15):153902)

Figure 7 - Magnetic system for direct observation and measurement of DNA packaging force on the phi29 DNA packaging motor.

5. We have also created and continue to expand a molecular toolkit of modified lipids and nucleic acids for motor re-engineering and for incorporation of motors in membranes. For example, we have devised a DNA oligonucleotide capable of switching the phi29 DNA packaging motor (J Am Chem Soc. 2008 130(52):17684-7)

We report a reversible switching of the biological activity of an RNA molecule, packaging RNA (pRNA), which is a central component of the DNA packaging motor of bacteriophage phi29. The switching mechanism contains two components: (1) inhibition of pRNA by a short antisense DNA (asDNA) that can bind to the 3′ end of the pRNA and inactivate the packaging motor; and (2) reactivation of pRNA by isothermal removal of asDNA from pRNA through a strand displacement strategy. The switching process can be repeated for multiple cycles and has been demonstrated by gel electrophoresis and a virion assembly assay.

 

Figure 9 - Reversible switching of the phi29 motor function measured as a plaque formation.

Figure 9 - Reversible switching of the phi29 motor function measured as a plaque formation.

6. We have successfully incorporated phi29 motor components into nanoscale array structures including ordered arrays of phi29 connectors on a lipid monolayer substrate and with a unique nanoporous anodic alumina template. (Biomedical Microdevices, 11(1) 135-142)

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Pathway to Medicine

Our main PtM focus was on the Challenge 1 goal of packaging DNA within a liposomal carrier for gene therapy applications. Since the impact of such a carrier system would be broad, we initially chose to address the question of specific application once the packaging limitations and pharmacokinetic properties of the system were elucidated. While demonstration of Phi29 motor function was originally a Challenge 1, Year 3-4 goal, the primary purpose of the move to Cincinnati was in fact to provide greater access to clinical research that might eventually provide application for the Phi29 motor. Since that move, the PI and co-investigators have established an engaged Clinical Advisory Board (CAB). In close consultation with our CAB, we have identified a series of clinically relevant research projects with expert collaborators for specific disease. These indications provide an excellent ‘fit’ for the Phi29 technology in that RNA molecule-based therapeutic research with appropriate animal model systems are already underway in the laboratories of the clinical collaborators. We recognize that human therapeutic application for the Phi29 motor will not be a short term reality; however, quarterly meetings with our CAB will guide and evolve our clinically relevant research efforts. It is our contention that attempting to explore too many pathways at once will only complicate the desired results of expedited identification of clinical applicability. Thus, the Executive Committee has gathered information on especially promising medical focus areas that would benefit from this technology.

Figure 11 - Potential use of phi29 pRNA hexamers as polyvalent delivery vectors.

Clinical Advisory Board
 

  • Greg Grabowski, MD
  • William S. Ball, MD
  • John J. Rossi, Ph.D.
  • Shuk-Mei Ho, Ph.D.
  • Susan Clare, MD, Ph.D.
  • Punam Malik, MD
  • Ruth O'Regan, MD
  • John Perentesis, MD
  • George Sledge, MD
  • Peter Stambrook, Ph.D.
     

Executive Committee
 

  • Peixuan Guo, Ph.D.
  • Carlo Montemagno, Ph.D.
  • David Thompson, Ph.D.
  • Rashid Bashir, Ph.D.
  • Greg Grabowski, MD
  • John J. Rossi, Ph.D.
  • Shuk-Mei Ho, Ph.D
  • William Ball
     

References

Current NDC Supported Papers
 

  1. Zhang, H., Shu, D., Browne, M., Guo, P. 2009. Construction of a laser combiner for dual fluorescent single molecule imaging of pRNA of phi29 DNA packaging motor. Biomedical Microdevices. 2009 Oct 7. [Epub ahead of print] (PMID: 19809878)
  2. Wendell, D., Jing, P., Geng, J., Subramaniam, V., Lee, T. J., Montemagno, C. D. & Guo P. 2009. Translocation of double stranded DNA through membrane adapted phi29 motor protein nanopore. Nature Nanotechnology, 4(11): 695-780. (PMID: 19893523)
  3. Lee, T.J., Zhang, H., Chang, C., Savran, C, Guo, P. 2009. Engineering of the Fluorescent-Energy-Conversion Arm of Phi29 DNA Packaging Motor for Single-Molecule Studies. Small, 2009 Nov;5(21):2453-9 (PMID: 19743427)
  4. Xiao F., Cai Y., Wang, J.C., Green D., Cheng R.H., Demeler, B.,and Guo, P. 2009. Adjustable Ellipsoid Nanoparticles Assembled from Re-engineered Connectors of the Bacteriophage Phi29 DNA Packaging Motor. ACS Nano. 3(8):2163-70.
  5. Zhang, F.M., Su, Y., Guo, S., Yuan, J., Lima, T., Liu, J., Guo, P., Yang, D. 2009. Targeted delivery of anti-coxsackievirus siRNAs using ligand-conjugated packaging RNAs. Antiviral Research83: 307–316.
  6. Li, L., Liu, J., Diao, Z., Shu, D., Guo, P., Shen, G. 2009. Evaluation of specific delivery of chimeric phi29 pRNA/siRNA nanoparticles to multiple tumor cells. Mol. BioSyst., 5(11): 1361-1368.
  7. Lee, T., Schwartz, C., and Guo,P. 2009. Construction of Bacteriophage Phi29 DNA Packaging Motor and Its Applications in Nanotechnology and Therapy. Annals of Biomedical Engineering 27(10): 2064-2081.
  8. Zhang, H., Shu, D., Browne, M., Guo, P. 2009. Approaches for stoichiometry and distance measurement within nanometer bio-complex by dual-channel single molecule imaging. Proceedings of IEEE-NIH Life Science Systems and Applications Workshop 2009 (LISSA 2009). 124-127. (NIHMSID # 130790)
  9. Venkatesan, M., Dorvel, B., Yemenicioglu, S., and Bashir, R. 2009. Highly sensitive, mechanically stable nanopore sensors for DNA analysis. Advanced Materials, 21(27): 2771. (NIHMSID #131723)
  10. Moon,J., Akin, D., Xuan,Y., Ye, P., Guo, P. and Bashir,R. 2009. Capture and alignment of phi29 viral particles in sub-40 nanometer porous alumina membranes. Biomedical Microdevices, 11(1) 135-142. (NIHMSID #131732)
  11. Xiao, F., Sun, J., Coban, O., Schoen, P., Guo, P. 2009. Fabrication of Massive sheets of single layer patterned arrays using reengineered phi29 motor dodecamer. ACS Nano, 3: 100–107. (PMCID: PMC2651733)
  12. Ko, S.H., Chen, Y., Shu, D., Guo, P., and Mao, C. 2008. Reversible switching of pRNA activity on the DNA packaging motor of bacteriophage phi29. J. Am. Chem. Soc., 130(52): 17684-17687. (NIHMS86643)
  13. Chang, C., Zhang, H., Shu, D. Guo, P., Savran, C. 2008. Bright-field analysis of Φ29 DNA packaging motor using a magneto-mechanical system. Appl. Phys. Lett., 93(15):153902. (PMCID: PMC2684695 [Available on 2009/10/13])
  14. Xiao, F., Zhang, H., and Guo, P. 2008. Novel mechanism of hexamer ring assembly in protein/RNA interactions revealed by single molecule imaging. Nucleic Acids Res. 36:6620-6632. (PMCID: PMC2582624)
  15. Lee, T.J., Zhang, H., Liang, D., and Guo, P. 2008. Strand and nucleotide-dependent ATPase activity of gp16 of bacterial virus phi29 DNA packaging motor. Virology, 380, 69-74. (PMCID: PMC2585381 [Available on 2009/10/10])
  16. Zhang, C., Su, M., He, Y., Fang, P., Ribbe, A.E., Jiang, W., and Mao, C., 2008. Conformational flexibility facilitates self-assembly of complex DNA nanostructures. Proc. Natl. Acad. Soci. USA., 105(31)10665-10669. (PMCID: PMC2504817)
  17. Venkatesan, B.M., Dorvel,B., Polans,J., and Bashir, R. 2008. Fabrication of low stress, low capacitance gAluminum Oxide Nanopores for the electronic detection of biomolecules, Proceedings of Fourth Focused Workshop on Electronic Recognition of Bio-Molecules, 4, Sept.10-12, 2008. (NIHMSID # 131821)
  18. Wang, C.Y., Miyazaki, N., Yamashita, T., Higashiura, A., Nakagawa, A., Li, T.C., Takeda, N., Xing, L., Hjalmarsson, E., Friberg, C., et al. 2008. Crystallization and preliminary X-ray diffraction analysis of recombinant hepatitis E virus-like particle. Acta Crystallogr Sect F Struct Biol Cryst Commun. 64(Pt4):318-22. (PMCID: PMC2374242)
  19. Fang, Y., Shu, D., Xiao, F., Guo, P., and Qin, Z.P. 2008. Modular assembly of chimeric phi29 packaging RNAs that support DNA packaging. Biochemical and Biophysical Research Communications, 371, 589-594. (PMCID: PMC2504005 [Available on 2009/08/08])
  20. Guo,P. and Lee,T.J. 2007. Viral nanomotors for packaging of dsDNA and dsRNA. Mol. Microbiol. 64, 886-903. ( PMCID: PMC2366019)
     

Contact Information

Web site:

http://www.eng.uc.edu/nanomedicine Exit Disclaimer

Principal Investigator:

Peixuan Guo, Ph. D.
Dane and Mary Louise Miller Endowed Chair in Biomedical Engineering
Dept of Biomedical Engineering
The Vontz Center for Molecular Studies
3125 Eden Avenue, Room 1301
University of Cincinnati/College of Engineering/College of Medicine
(513) 558-0041
guopn@uc.edu

Or Dr. Anne Vonderheide
(513) 556-5568
pawleca@uc.edu

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