Phi29 DNA-Packaging Motor for Nanomedicine
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, P Guo 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 as a mechanism 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 selective detection of biomarkers, diseased cells, or pathogens.
Introduction
Nature 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.
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- Figure 1. Schematic of the phi29 RNA nanomotor
NDC Goals
The goal of this Nanomedicine Development Center (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. The Center will create biologically compatible membranes and arrays with embedded and active phi29 DNA-packaging motors for applications in medicine. Our NDC will create a hybrid system that combines the best features of the biological motor, re-engineered motor components to provide specific functionalities, and synthetic delivery vehicles for drugs that have already achieved clinical success. Developed re-engineered motors will also be applied in various array formats for diagnostics applications.
Three challenges to development of clinically viable applications for the phi29 DNA-packaging motor are elaborated:
Challenge 1: Re-Engineering the Phi29 Motor for Incorporation into Lipid or Polymer Bilayers.
A vital element of this challenge is the successful incorporation of the phi29 motor into bilayer membranes allowing therapeutic cargo (DNA, RNA or drugs) to be packaged within a liposome in a reliable and predictable manner. This objective will also be achieved with control over directionality so that the motor will be embedded in the membrane with the same topological orientation that exists in the native bacteriophage. Although conceptually similar to the native activity of packaging DNA, substantial engineering and manipulation of Phi29 motor components is required for application of motor structure and function in medicine. Motor components must be made resistant to degradation and compatible with complex hydrophobic membrane environments in addition to directing and preserving motor pumping activity. Working nanomotor studded liposomes or polysomes must also be examined for toxicology and pharmacokinetics and optimized for medical application.
Challenge 2: Mechanistic Studies of the Re-Engineered Motor.
A significant challenge in the assembly of a nanoscale machines is precise, sensitive and consistent measurement of desired (and undesired) results. The study includes the use of unique properties of phi29 motor components to build nanomachines and arrays. It involves optical and mechanical manipulation to regulate the re-engineered motor, with control of nanomotor orientation and switching of motor activity. The capabilities of the research team will be directed to exhaustive and diverse performance testing of re-engineered motors.
Challenge 3: Development of active nanomotor arrays that enable drug delivery and diagnostics.
Higher order structures will be designed and built to utilize re-engineered motors for medical applications. These include nanoscale porous membranes to support motor assembly and nucleic acid lattices for specific functional activities. Motor components will be employed to produce active and passive motor array architectures for selective detection of biomarkers, diseased cells or pathogens.
Studies on the bacteriophage phi29 DNA packaging motor have been carried out in several laboratories for many years. However, there have been no prior attempts to adapt the phi29 motor to the lipid membrane or other matrix substrates for therapeutic applications. This is a high-risk project that would not be fundable via the traditional mechanisms. The uniqueness of this proposal is to construct a motor that will be embedded in membrane sheets or vesicles (e.g., liposomes or polymersomes). Our NDC aims to apply deep knowledge of phi29 motor function to a membrane-adapted form of the motor. This is a novel, unique and unprecedented approach. Creation of an engineered membrane system that incorporates bacterial virus nanomotors and is predictable, stable and robust in eukaryotic systems is an ambitious goal. To include in such a program active and functional nanomotors presents an even more significant challenge. The scope and complexity of constructing the new generation of viral DNA packaging motor adapted to membranes or other substrates as well as mechanistic studies of the lipid adapted nanomotor for the treatment and diagnosis of cancer, infectious and genetic diseases, requires a multidisciplinary network of investigators. This network includes molecular biologists, synthetic chemists, lipid chemists, biomedical engineers, computational scientists, 3-D modeling experts, electrical engineers, imaging scientists, tructural biologists, biophysicists, biotechnologists, and clinical physicians, the majority of whom were already engaged in well-established, successful collaborations. Previous efforts by members of the assembled team examined the biological, physical, mathematical foundations of the phi29 DNA translocation motor. With an expanded multidisciplinary representation, the Center team can integrate previous findings with the engineering principles underlying the motor's structure and function. The NDC multidisciplinary team approach assembles expertise to meet these goals, including for example, deep experience with innovative liposomal delivery vessels and nano-engineered array materials that will provide the substrates for incorporation of re-engineered nanomotors. The distinct advantage of this integrative and collaborative effort will be an understanding of the intricate interplay between structural, mechanical, and chemical forces that will enable construction of a nanomechanical device prototype for nanomedicine experiments.
Accomplishments
Progress has been made in each challenge during the first year of operation of the NDC. For example, 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 (Fig. 2).
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- Figure 2. Magnetic system for direct observation and measurement of DNA packaging force on the phi29 DNA packaging motor
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 (Fig. 3).
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- Figure 3. Reversible switching on the phi29 motor function measured as a plaque formation with a small interfering DNA targeting to motor component
Of particular significance 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 (Fig. 4). Progress has already come with incorporation of phi29 motor components in vesicle structures (fig. 5).
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- Figure 4. Three phi29 array structures. A) Field electron micrograph of viral particles attached to nanospores of anodic alumina. B) Atomic force microscope image of a single layer connector array. C) TEM image of single layer connector array constructed with new nanotechnology.
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- Figure 5. Left: Fluorescently labeled and modified phi29 connector inserted into the membranes of a liposome vesicle. Right: Uniform fluorescence from vesicles incubated with free dye (control.)
Our studies began in the context of our deep experience with of 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.
Future Applications
The phi29 DNA packaging motor has general relevance to a wide variety of biochemical and biophysical processes. The information derived from the NDC will guide other adaptations of biological motors for nanomedical applications. It is the intent of our Center to uncover the fundamental metrics and design concepts in a prototypical DNA packaging motor and to extend to other ATP-driven packaging motors. Physical testing and in silico computational modeling of the motor and nanomedical agents developed by the Center will reveal the optimal parameters for materials, physical design configurations, and real-world (in vivo) structural durability, including minimal or maximal "operating limits". This work will provide information for electrical, mechanical and materials engineers interested in nanotechnology to understand the principles of magnetic force fields, rates of rotation, physical configurations, energy efficiency, torque generation, biomachine durability, operating limits; thermal, structural, and magnetic properties, and resistance to harsh environments. Many future applications of the phi29 motor system can be anticipated.
- These studies can serve as a model for: (a) the study of viral DNA packaging and design of new antiviral strategies; (b) RNA/RNA and stable RNA multimer formation; and (c) RNA/protein interaction via understanding of the pRNA/connector complex.
- The phi29 motor functions in a manner similar to other nucleic acid processing protein or enzymes that play a role in DNA or RNA riding or sliding motor, including helicases, terminator Rho, and other DNA binding factors.
- Phi29 DNA translocation has similarities to DNA traversing a protein barrier and other macromolecular transport across cell membranes.
- Application of the DNA-packaging motor and its components to nanotechnology, where the motor and pRNA dimers, trimers and hexamers could be used as mechanical nano-parts.
- Viable and effective biomimetics nanodevices will serve as models for the application of other biomachines and for self-assembled structures as templates in nanomedicine applications.
- The construction and modification of arrays of pRNA and motor components can provide templates to build patterned superstructures. Assembled tetragonal or hexagonal arrays of phi29 motor components can be employed to produce metal replicas that will serve as diagnostic and detection chips.
- Stable and biocompatible pRNA scaffolding can be constructed for wound repair.
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- Figure 7. Targeting VEGF to inhibit neovascularization.
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- The phi29 motor system serves as a model for the study of energy transduction.
- Introduction of photochemical switches and other external control elements/nano-components to the connector-pRNA will bridge the gap between biological and the engineering elements of nanotechnology.
- Use of multivalent therapeutic pRNA nanoparticles in patient clinical trials for the treatment of cancers, genetic diseases, and viral infections, including avian influenza virus, hepatitis B and HIV-AIDS (e.g., Fig. 6). The use of such non-antigenic 20-40 nm particles precludes induction of an antibody immune response and could provide long-term treatment options for chronic diseases.
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- Figure 6. Potential use of phi29 pRNA hexamers as polyvalent delivery vectors. Six positions are available to carry specific therapeutic and diagnostic payloads.
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- Inhibition of neovascularization is another potential application. A major target in malignant, cardiovascular, ocular and infectious diseases is the selective blockade of pathological tissues that are undergoing neovascularization. VEGF is one biomarker that has been actively investigated as a potential target for anti-angiogenic therapy. We will develop a complementary approach based on delivery of siRNA with the phi29 motor that targets the knockdown of aminopeptidase N (APN) in tumor vasculature (Fig.7). APN, a membrane-bound metalloprotease, has recently been identified as a surface marker in myeloid cells. APN knockout mice have also been shown to display impaired angiogenic response under pathological conditions. Our long term goal is to develop VEGF-targeted liposomes carrying APN siRNA that has been packaged using the phi29 motor. The advantages of this system are 1) the capacity to utilize a polyvalent approach to VEGF targeting on the surface of fluid phase liposomes to increase the efficiency of surveillance and binding to tissues that are actively undergoing neovascularization; 2) the ability to passivate siRNA toward immune recognition of CpG motifs in the nucleic acid therapeutic, thereby enabling multiple injections of the vector without stimulating it's rapid clearance as is known to occur for unpackaged, non-human nucleic acid constructs; and 3) the capability of producing multifunctional antitumor agents that can be programmed to package APN siRNA and doxorubicin or other cytotoxic compounds in a novel, two-agent approach that can be adapted for a variety of tumor targets.
We have begun to think in terms of motor therapeutic delivery capabilities in head and neck cancer (Guo et al, 2005). Each pRNA molecule will be selectively engineered with a payload relevant for therapy. With this system, we can take advantage of increasingly confident data describing disease-specific gene expression; one or more of the pRNA molecules can carry small interfering siRNA, aptamer, or ribozyme against such disease specific genes. Combinatorial therapy is increasingly used for complex diseases, especially cancer, and the envisioned vector provides a polyvalent structure that can carry different types of therapy including traditional small molecule drugs and therapeutic moieties. The vector may include an imaging agent attached to another of the hexamer pRNAs to enable therapy to be monitored and to ensure that the vector reaches the intended target in vivo (Fig. 6). The hexameric vector can also be programmed to 'work' once it reaches its target for example by including on one pRNA a chemical to release the vector from host cell endosomes.
References
Background
- Guo S, Tschammer N, Mohammed S, Guo P., "Specific Delivery of Therapeutic RNAs to Cancer Cells via the Dimerization Mechanism of phi29 Motor pRNA.", Human Gene Therapy, 2005:16(9):1097-1109.
- Ramachandran S, Ernst KH, Bachand GD, Vogel V and Hess H., "Selective Loading of Kinesin-Powered Molecular Shuttles with Protein Cargo and its Application to Biosensing", Small, 2006:2(3):330 - 334.
- Soong RK, Bachand GD, Neves HP, Olkhovets AG, Craighead HG, Montemagno CD., "Powering an inorganic nanodevice with a biomolecular motor."Science, 2000:290(5496):1555-1558.
- Guo, P., S. Erickson, and D. Anderson. "A small viral RNA is required for in vitro packaging of bacteriophage phi29 DNA."Science, 1987; 236(4802):690-694.
- Guo P, Lee TJ. "Viral nanomotors for packaging of dsDNA and dsRNA". Mol Microbiol. 2007;64(4):886-903.
Contact Information
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@ucmail.uc.edu
Website
Co-investigators
David Thompson, Ph.D.
Professor of Chemistry
Purdue University
560 Oval Drive
West Lafayette, IN 47907
765-494-0386
davethom@purdue.edu
Website
Rashid Bashir, Ph.D.
Professor of Electrical and Computer Engineering
Purdue University
Birck Nanotechnology Center
1205 West State Street
West Lafayette, Indiana 47907-2057
765 49-66229
bashir@ecn.purdue.edu
Website
Carlo Montemagno, Ph.D.
Dean, College of Engineering
University of Cincinnati
PO Box 210018
Cincinnati, OH 45221-0018
513-556-2933
carlo.montemagno@uc.edu
Website
