2008 Progress Report – Executive Summary
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:
- Re-engineering the phi29 motor for incorporation into
lipid, polymer or cell membranes
- Mechanistic studies of the re-engineered membrane adapted
motor
- 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.
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