Phi29 DNA-Packaging Motor for Nanomedicine
Nature has achieved tremendous design optimization and efficiency to perform work in biological systems. Deep and broad fundamental research over the past 20 years has led to a good understanding of the molecular mechanisms of Phi29 DNA-packaging motor activity. For example, the Phi29 connector is a self-assembled 12- fold symmetrical ring of gp10 proteins. The resulting 20-nanometer cylindrical structure contains a 3.6- nanometer central channel, which makes it an ideal candidate as a building block for nanotechnology and nanomedicine applications. Six copies of the motor pRNA (packaging-RNA) form a 7.5-nanometer ring sheath onto the 6.8-nanometer narrow end of the connector. The goal of this Nanomedicine Development Center (NDC) is to create biologically compatible lipid or polymer membranes and metal or silicon arrays with embedded phi29 DNA-packaging motors for applications in medicine. To achieve this goal, three important research challenges are elaborated:
1. Re-engineering the phi29 motor for incorporation into lipid, polymer or cell membranes
2. Mechanistic studies of the re-engineered membrane adapted motor
3. Active nanomotor/metal and silicon arrays that enable drug delivery and diagnostics
Challenge 1: Re-Engineering the Phi29 Nanomotor for Incorporation into Lipid, Polymer or Cell Membranes
The lipid bilayer membrane is an appropriate vehicle to host and direct nanomotor activity for diagnostic or therapeutic activity in humans. The emerging promise is that liposomes of various types can be generated for different applications such as to deliver specific therapeutic cargo or for targeting to specific cells and organs. Activities in the first challenge are directed at co-opting the simplicity and efficiency of design of the phi29 motor to provide effective machinery for movement and transport of cargo in human cell and tissue systems. We explore the feasibility of incorporating the native Phi 29 connector into the membrane of liposomes, polymersomes, and sheets composed of lipid or polymers, which will enable both passive and active delivery of drugs and/or DNA to the targeted cells. For passive membrane-bound motors, the connector will be incorporated into the membrane and serve only as a pore. The main concern is that the alternating hydrophobicity and hydrophilicity of the three-layered motor connector structure is unfavorable to its insertion into the membrane of liposomes. Initial studies during the first 16 months of this center with several approaches to incorporate the phi29 nanomotor into liposomes and polymers are beginning to demonstrate the feasibility. Activities in challenge 1 are already underway to establish the best strategies to reengineer the connector to attach or insert the connector to liposomes, lipid sheets, or polysomes by chemical conjugation and bottom-up assembly.
Activity in this challenge includes development of strategies to incorporate active and functional phi29 motors into membranes. Gaining control over the orientation of motor assembly and incorporation will be a major goal. The stabilization of both protein and nucleic acid motor components in the heterologous lipid or polymer environment will be required. Research will be undertaken to develop methods for encapsulation of therapeutic and diagnostic cargo into the motor studded liposomes. These procedures must eventually meet clinical standards and therefore must be robust, reproducible and efficient. Ongoing work will focus on methods for tagging motor-studded and loaded liposomes to track these materials in cell culture and ultimately in vivo as well. A key issue with our approach to the incorporation of this nanomotor into liposomes is the potential toxicity and immune response associated with these constructions as therapeutics. We expect the materials to be very well tolerated based upon the excellent track record for liposomes in the clinic. However, this must be rigorously tested in cell and animal systems. Similarly, pharmacokinetic studies will be carried out to evaluate adsorption, distribution, metabolism and excretion of these materials as required for characterization of therapeutics.
Challenge 2: Mechanistic Studies of the Re-Engineered Membrane Adapted Motor
To effectively employ the phi29 DNA packaging motor for therapeutic and diagnostic purposes as proposed in Challenge 1, we must better understand the engineering principles underlying its design and function. Substantial molecular characterization of the native motor system has been obtained in the laboratory of the PI and many others. Previous extensive studies on the mechanism of phi29 DNA packaging motor from variety of labs focused on the functional motor or motor components in the native or similar environment. However, a deeper and system-based understanding of the reengineered and membrane adapted motor will be required to co-opt and even to improve the motor for Nanomedicine applications. Our NDC center will take very different approaches. The focus will be at the interface between bio and non-biomaterials, and the function of the motor and components in non-native environment. For example, we will elucidate the structure and function of the motor or the motor components incorporated into exotic environments such as on silicon arrays, aluminum alloys or other metals and in synthetic lipid bilayers or polymers. Establishing the function of the membraneadapted motor will be our major task. The engineers, physicists and other NDC team investigators are currently working to obtain this deep understanding of principles for motor reengineering. This team has the resources and capabilities to then re-engineer motor components and to obtain the artificial motor activity for specific applications. During the first sixteen months of studies we have achieved substantial amount of data showing that phi29 nanomotor or motor components could function in an exotic environment with special design, conjugation, engineering, and modifications. For example, some motor components such as the motor connector, the motor pRNA, as well as the motor DNA packaging protein gp16 have retained certain biological properties after being engineered. These properties include correct folding and appropriate structure in interaction with other motor components in the assembly of functional motor or into useful other structures with desired conformation.
Methodologies that promote better control of the phi29 motor function have been employed. These include ultra-high resolution imaging, specially formulated and equipped analytical centrifugation, ultra-fast photography, a customized single molecule dual-view imaging system and special AFM technology. We have also utilized special methods for the production of arrays with lipids, silicon and aluminum. Motor components are evaluated with cryo scanning probe tunnel (STEM) and variable vacuum electron microscopy to achieve more detailed structural and functional characterization of the modified motors and motor components to elucidate relationships between structure and function in membrane adaptation.
Physical methods to measure forces in the interaction of motor components and lipid or polymer membrane at the nanoscale are in development and application by NDC investigators. Various beads and cantilever based approaches already hold promise for accurate and facile monitoring of motor activity in membrane transport. Lessons learned from those approaches will be key stepping stones for gaining control to assemble functional motors that incorporate into the membrane or embed in a foreign environment. A first step will be to apply these findings to control motor orientation upon reengineering or incorporation, especially in heterologous systems such as the liposome. The measurements made at this scale will be extended with molecular modeling, which will also lead to new hypotheses concerning nanomotor function.
Importantly, it is anticipated that this mechanistic understanding of motor function will directly suggest alternative approaches to DNA nanoparticle assembly. With components of the motor understood at the molecular and atomic scale, it will be crucial to formulate chimeric motors with ‘bullet proof’ components to provide in vivo durability and therapeutic impact. At the same time, specific motor functions can be exclusively exploited with re-engineered motor constructs. The developed re-engineered motors will, of course, require characterization with respect to activity, biocompatibility, durability, toxicity and biodistribution as described above for challenge 1.
Challenge 3: Active Nanomotor/Metal, DNA or Silicon Arrays that Enable Drug Delivery and Diagnostics
Nature has created elegant nanomachines and ordered nanostructures. The novelty and ingenious design of such biological machines and structures have helped inspire the development of biomimetics by using such components to build arrays with ultra small structures. Much current research is being devoted to make these machines applicable outside of their native environment, with an intention to use them in nanotechnological and medical applications such as drug delivery, identification of pathogens or diseases, design of MEMS, driving of molecular sorters, and the building of molecular sensors and complex actuators. One important area in nanotechonology lies in the interface between biological macromolecule and material sciences. One of the first stages in the integration of biological components into nanotechnology is to produce hybrids of biological, chemical, and other synthetic materials. This will allow us to translate biomolecular processes into controlled and directed operations with specific recognition functions. The presence of multiple subunits in each phi29 motor component can be used to amplify detection or target signal in these applications. For example, six copies of pRNA and twelve copies of connector protein gp10 will facilitate the identification of multiple pathogens or genes. During the first sixteen months of our NDC activity, we have created and assembled a variety of arrays with the distance between each unit within several to tens of nanometers. Furthermore, various arrays with hybridization of Phi29 motor components and organic or metallic materials, such as nanoporous anodic aluminum oxide, have been produced.