Nanomedicine Center for Nucleoprotein Machines
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.