2008 Progress Report – Executive Summary
Nucleoprotein machines manage the business of information
storage and transfer for the cell, including DNA replication
and repair, RNA synthesis, and protein translation. The science
and engineering focus of this center is on developing tools
and concepts to better understand nucleoprotein machines,
using as a prototype a natural machine that repairs DNA double-strand
breaks. The center is combining bright photostable probes,
super-resolution microscopy, and other physical and computational
tools to investigate assembly, disassembly, and control of
the DNA break-repair machine. Inspired by a natural process
of V(D)J recombination, which uses the double-strand break
repair machine to insert, delete, and alter DNA sequences
at specific sites, the center also hopes to create engineered
nanomachines to provide genetic cures for common human diseases.
Such machines would be most applicable to diseases that are
reasonably common, life-threatening, caused by a single-gene
defect, and where correction of the defect in even a fraction
of cells would provide clinical efficacy. Examples of diseases
that meet these criteria are hemoglobinopathies, such as
sickle cell disease, and triplet-repeat expansion neurodegenerative
diseases, such as Huntington’s disease.
Consistent with the vision of NIH Roadmap Initiative in Nanomedicine,
the long-term goals of our NDC are bold
and ambitious: we hope to obtain a general understanding
of engineering design principles of nucleoprotein machines
that carry out templated copying of nucleic acids, mediate
genetic recombination, and catalyze RNA processing; we hope
to establish the ability to precisely modify the information
stored in DNA and RNA, thus providing novel therapeutic strategies
for a wide range of human diseases, including sickle cell
disease. We realize that achieving this ability may take
a few decades and will require significant development and
clinical resources; however, our initial work in developing
a Pathway to Medicine (PtM) will serve as a test bed for
such a vision.
The major short-term goals of our NDC are
to develop novel fluorescent probes, orthogonal tagging strategies,
new imaging methods and computational tools, and to characterize
the dynamics of a model nanomachine, the nonhomologous
end joining (NHEJ) complex that repairs DNA double strand
breaks. The NHEJ complex is relatively simple - it has fewer
than 10 core components. It is clinically relevant since
it controls the response of tumor cells to radiation therapy.
Even a modest ability to redirect or inhibit this repair
pathway in tumor cells would dramatically increase patient
survival in diseases such as lung cancer. Importantly, NHEJ
occurs within self-organizing structures, or “repair foci”
that are amenable to live-cell visualization using
technologies being developed in our NDC. NHEJ is also relevant
to the long-term goals of the Nanomedicine initiative. The
adaptive immune response, which is the only physiological
process in humans that involves modification of somatic DNA,
uses the core NHEJ machinery. A better understanding of the
NHEJ machine, including its structure-function relations
and associated signaling and control loops, will be an important
step toward the long-term goal of manipulating DNA and RNA
at will.
In
achieving our short-term and long-term goals, it is critical
to visualize the dynamics of assembly and disassembly of
single NHEJ complexes in the nuclei of living cells, which
is far beyond the limits of existing technology. Through
a close collaboration of biologists, bioengineers, chemists,
and computational scientists in our NDC, over the last 9
months, we have been developing: (1) small, bright, stable,
and biocompatible fluorescent probes; (2) orthogonal tagging
strategies to tag components of the NHEJ; (3) expression
systems for mammalian and yeast cells to label NHEJ components
with fluorescent proteins; (4) single-molecule measurement
tools to validate the NHEJ formation in vitro; (5)
live-cell imaging methods for tracking proteins and nucleic
acids in cell nucleus; (6) electron microscopy methods for
high-resolution structure of protein complexes; (7) computational
tools to simulate nanomachine formation, to interpret and
quantify the experimental data, aiming to identify engineering
properties of the nanomachines.
During the first 12 months (3/1/07-2/29/08) of our NDC, we
have carried out research with the following Specific
Aims that are closed related and inter-dependent.
The tasks for specific laboratories involved in our NDC are
shown in Chart 1.
Aim 1. Develop protein tagging strategies and
fluorescence probes for nanomachine targeting. We
will design, synthesize and validate new optical
imaging probes that are small (4-6 nm), biocompatible,
photostable and potentially activatable, and test
orthogonal protein targeting/tagging strategies to
label 4-6 components of a nanomachine with high specificity.
Aim 2. Decipher structure-function relationship
of components required for the core NHEJ reaction. We
will assemble a core NHEJ complex in vitro from
purified components and characterize it by singlemolecule
approaches. We will measure rates of assembly and
disassembly, characterize the complex formed, and
measure its resistance to disruption by tensional
and torsional forces. We will also extend these in
vitro studies to V(D)J recombination, a somatic
recombination process that uses the core NHEJ machine.
Aim 3. Characterize the dynamics of nanomachine
assembly and disassembly in the context of repair
foci. We will induce DSBs in a controlled
and synchronous manner in vivo. We will
use optical imaging methods with improved sensitivity
and spatial resolution to characterize assembly and
disassembly of the repair nanomachine on a chromatin
substrate in living cells.
Aim 4. Determine the dimensions and structure
of repair foci at high resolution in fixed cells. We
will measure the dimensions of repair foci as a function
of time following DSB induction, and investigate
their internal structure and composition using high
resolution methods applicable to fixed cells. We
will use electron cryo-microscopy, dual contrast
probes, and novel sample preparation methods.
Aim 5. Establish the engineering design principles
underlying DNA double-strand break repair. We
will elucidate the signaling-response-feedback-control
loops that connect different aspects of the DSB response
and develop a mathematical model for quantifying
the reaction kinetics, the growth and disappearance
of repair foci, and the amplification of DNA damage
signals.
A major near-term goal of our NDC is to track the assembly
and disassembly of single NHEJ complexes in living cells.
To achieve this goal, over the last 12 months we developed
methods for labeling NHEJ components in both mammalian and
yeast cells. We have developed a new protein labeling platform
based on lipoic acid ligase (LplA) from E. coli that
is small, rapid, and highly specific relative to the BirA
labeling strategies. We are currently using this methodology
to perform protein crosslinking experiments in vitro and
inside living cells. We have been developing new, small (~
1 nm), FRET-based protein probes for intracellular targeting.
These probes are activataqble, i.e., they become fluorescent
only when bind to the target protein, thus reducing the background
signal. They use organic dyes and thus can be readily color-multiplexed.
We have also developed a new class of ultrasmall quantum
dots (QDs) and nanogold probes for intracellular imaging.
The QDs surfaces are coated with multivalent copolymer ligands
that are able to penetrate cell membranes. Although rapidly
internalized by endocytosis, the surface coating disrupts
endosomes releasing the QDs into the cytoplasm. Using an
entirely different approach, we have also made progress in
synthesizing highly fluorescent and water-soluble metal nanoclusters
based on the use of multivalent coordinating polymers. We
have successfully tagged the Ku70/Ku80 complex (mCherry-Ku),
XRCC4 (XRCC4-YFP), and a synapsis factor, PSF•p54 dimer (GFP-PSF)
with fluorescent proteins mCherry, YFP and GFP, respectively.
We confirmed the expression of a correct-size polypeptide
by in vitro transcription-translation and confirmed
that fluorescent products could be expressed and correctly
localized in mammalian cells (Fig. 2). We found that the
tagged NHEJ proteins localized to the cell nucleus. We also
made improvements on fluorescence live-cell imaging methods
with multi-color capabilities, and instrumentation and techniques
of single-particle imaging in electron microscopy. Further,
experimental and analytic tools have been developed to characterize
the dynamic properties of NHEJ proteins in the absence of
DSBs, especially the low-affinity binding reactions.
Modern medicine – allopathic medicine – focuses on treating
symptoms, commonly through small-molecule enzyme inhibitors
and receptor agonists/antagonists and does not address underlying
genetic causes. Consistent with the vision of the Nanomedicine
Roadmap Initiative, we anticipate that, in the future, the
allopathic model can be replaced by therapies that directly
modify information contained in DNA and RNA. Therefore, a
major long-term goal of our NDC is to develop a gene correction
strategy through the Pathway to Medicine (PtM) efforts. Inspired
by the NHEJ and V(D)J recombination pathways, we will harness
DNA repair nanomachines to alter, insert, delete sequence
as a novel way to cure human diseases. We conceptualize the
medical application of our nanomedicine efforts, or our Pathway
to Medicine (PtM), in achieving this long-term goal in terms
of three elements: device, delivery,
and a specific disease target. We have identified
candidates for each. For the device, one
approach that we are exploring capitalizes on a recent discovery
that the recombination activator gene (Rag) proteins, which
normally initiate a specialized V(D)J recombination reaction
in developing lymphocytes, can be re-engineered to promote
site-specific homologous recombination. We will also explore
the use of Zinc Finger Nucleases as potential reagents to
create double-strand breaks at specific sites and then modify
the DNA sequence using NHEJ. For the delivery system,
we will develop an approach based on nanoparticles as a carrier,
which could have multiple functions, including delivery,
targeting and release functions. By tailoring the size, surface
coating and targeting ligand of the nanoparticle, optimal
performance in delivery will be achieved. For the disease,
we have selected sickle cell disease as our first disease
model. Upon success, we will make an effort to cure Huntington’s
disease using the novel gene correction method. These diseases
are reasonably common, and life-threatening. Importantly,
each is caused by a single-gene defect, and the disease mechanism
is such that 100% efficient correction of the disease genes
should not be required for clinical efficacy.
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