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Phi29 DNA-Packaging Motor for Nanomedicine

2010 Progress Report – Executive Summary

Figure 2 - A continuous current trace recording multiple connector insertions (-75mV) after connector-containing proteoliposomes were added. Inset: A proteoliposome reconstituted by FITC labeled connectors; Schematic showing the mechanism of connector insertion into planar lipid bilayer via vesicle fusion.The dsDNA bacterial virus phi29 packages its genomic DNA-gp3 into a procapsid during the last stage of maturation. This entropically unfavorable DNA packaging task is accomplished by an ATP-driven DNA-packaging motor geared by six pRNAs (packaging RNA). Individual pRNA molecules can assemble into dimers, trimers, and hexamers via hand-in-hand interactions between the two right and left interlocking loops. The unique features of the two independent domains and the two interlocking loops make phi29 pRNA a promising tool for bottom-up assembly, disease diagnosis, and delivery of multiple therapeutics with specific cell targeting capability. The central hub of the phi29 motor is a dodecameric connector composed of protein gp10, which forms a channel with 3.6 nm at its narrow end and 6 nm at its wider end. The channel serves as a path for dsDNA translocation. The 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 insights, 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.

Figure 2 - A continuous current trace recording multiple connector insertions (-75mV) after connector-containing proteoliposomes were added. Inset: A proteoliposome reconstituted by FITC labeled connectors; Schematic showing the mechanism of connector insertion into planar lipid bilayer via vesicle fusion.The goal of the NDC: Phi29 DNA-Packaging Motor for Nanomedicine is to bridge the knowledge gap at the bio- and nanomaterials interface by employing the well-studied bacterial phage phi29 DNA packaging motor as a model system for the development of nanomedicine applications with the help of a highly interdisciplinary team of researchers. During the course of study in the last 3.5 years, we have incorporated the connector channel into artificial lipid membranes, demonstrated DNA translocation through the connector channel by single channel conductance assays, and observed amazingly stable and uniform current in pico-ampere sensitivity of a single molecule. Currently, the well-studied artificial membrane-embedded pores have a limited pore size and hence can only translocate single-stranded DNA. The larger pore diameter of phi29 connector embedded in the membrane makes this system unique in translocating double- stranded DNA. This discovery immediately led to several new projects that are currently underway utilizing the membrane-embedded phi29 channels.
 The five most important advances in the center are:

1. We have confirmed the incorporation of the connector into lipid membranes by observing uniform and discrete current jumps when multiple connectors were fused into the bilayer sequentially at a constant potential (Nature Nanotech, 2009).
2. The connector channels in BLM are capable of sensing ion concentrations and identifying ion species.
3. The connector channels in the BLM are capable of translocating dsDNA and distinguishing linear dsDNA from circular dsDNA (Nature Nanotechnology, 2009).
4. We demonstrated that the phi29 motor pRNA nanoparticles constructed via RNA nanotechnology could be used to recognize and enter the HIV virus infected cells, and block the replication of the HIV virus in the cell.
5. Using ovarian cancer as a model, we revealed a synergetic effect of pRNA nanoparticles for co-targeting multiple locations by taking advantage of the multivalent nature of phi29 pRNA.
Figure 3 - (a) An illustration of double-stranded DNA translocation through the connector channel of phi29 DNA packaging motor. (b) Following the insertion of one connector, a burst of blockade events of channel conductance characteristic of DNA translocation were observed. Inset: One DNA blockade event magnified, showing the dwell time and current blockade amplitude.
Project Evolution and Advances

The major evolution is the new project in using nanopore of phi29 DNA-packaging motor for restoration of eye macular degeneration.

NDC Supported Publications in 2009

1. Wendell,D., Jing,P., Geng,J., Lee,T., Montemagno,C., and Guo,P. 2009. Double-stranded DNA translocation through a novel phi29 motor-based pore incorporated in lipid membranes. Nature Nanotech, 4, 765-772. PMCID: PMC2777743.
2. Guo P, Coban O, Snead N, Trebley J, Hoeprich S, Guo S, and Shu Y. 2010. neering RNA for Targeted siRNA Delivery and Medical Application. Adv Drug Deliv Rev. In press.
3. 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, 37, 2064-2081. PMCID in process.
4. Xiao,F., Cai,Y., Wang,J., Green,D., Cheng,R.H., Demeler,B., and Guo,P. 2009. Amendable and Adjustable Ellipsoid Nanoparticles Assembled from Re-engineered connector Proteins of bacteriophage phi29 DNA Packaging Motor. ACS Nano, 3, 2163-2170. PMCID: PMC2731514.
5. Lee,T.J., Chang,C., Savran,C, and Guo P. 2009. Reengineering and extension of the arms of phi29 DNA packaging motor of single molecule detection. Small, 5, 2453-2459. PMCID: PMC2837281.
6. Zhang H, Shu D, Browne M, and Guo P. 2010. Construction of a laser combiner for dual fluorescent single molecule imaging of pRNA of phi29 DNA-packaging motor. Biomedical Microdevices, 12, 97-106. PMCID: PMC2812712.
7. B. M. Venkatesan, A. B. Shah, J.-M. Zuo, R. Bashir, Advanced Functional Materials 2010, 9999, NA.
8. B. M. Venkatesan, B. Dorvel, S. Yemenicioglu, N. Watkins, I. Petrov, R. Bashir, Advanced Materials 2009, 21, 2771.

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