Nanomedicine Center for Nucleoprotein Machines
Targeted/Planned Enrollment Format Page. The mammalian cell nucleus is a membrane-bounded compartment that is filled with self-organizing, interconnected, nanometer-scale machines. Because these machines are made primarily of proteins and act on nucleic acid substrates, we call them nucleoprotein machines. Nucleoprotein machines carry out essential biological processes including synthesis, modification, and repair of DNA and RNA. As part of the NIH Nanomedicine Development Centers network, we have established a nanomedicine development center (NDC) focusing on DNA damage repair nanomachines, especially the nonhomologous end joining (NHEJ) complex that repairs DNA double strand breaks (DSBs). We have assembled an outstanding interdisciplinary team from eight institutions (Georgia Institute of Technology, Medical College of Georgia, Emory University, Cold Spring Harbour Laboratory, New York University, MIT, California Institute of Technology, and German Cancer Research Center), with significant expertise in cell and molecular biology of DNA damage repair, protein tagging and targeting, nanostructured probes, cryo-electron microscopy, single-cell imaging, quantitative image analysis and computational biology, and light microscopy instrumentation. Our short-term goal is to develop innovative technologies to visualize the assembly, function, and disassembly of individual nucleoprotein machines in their natural milieu. Our long-term vision is to provide genetic cures for common human diseases based on the ability to manipulate the somatic human genome using Nanomedicine.
DNA damage repair is vitally important to human health, as both normal metabolic activities and environmental factors can cause DNA damage, resulting in as many as 100,000 individual molecular lesions per cell per day. If allowed to accumulate without repair, these lesions interfere with gene transcription and replication, leading to premature aging, apoptosis, or unregulated cell division. We chose to focus on the NHEJ complex because: (1) the NHEJ machine is simple compared with most other nucleoprotein machines – it has fewer than 10 core components; (2) NHEJ occurs within self-organizing structures, or “foci”, that are amenable to live-cell visualization; (3) assembly of the NHEJ machine can be induced by a single event – one DSB at a defined genomic site. NHEJ is also very relevant to our long-term goal of manipulating DNA and RNA at the molecular level since, in cells of the immune system, this pathway performs precise combinatorial joining of germ line DNA segments to create novel antigen receptor in a process known as V(D)J recombination.
Significant technology development challenges exist in characterizing the dynamics and structurefunction relationships of nucleoprotein machines. Most biochemical and cell biological approaches measure average behavior of an ensemble of molecules. They rely on the untested and implausible assumption that each nucleoprotein machine of a given class is uniform in its composition and behavior. Therefore, it is essential to develop innovative technologies to measure the dynamic behavior of individual nucleoprotein PHS 398/2590 (Rev. 09/04, Reissued 4/2006) Page 3 Continuation Format Page machines in living cells, and to obtain a quantitative description of nanomachines in engineering terms. Specifically, to characterize the NHEJ complex, we aim to: (1) tag 4-6 of NHEJ components using the probes and orthogonal tagging strategies developed at our NDC; (2) validate the activity of the tagged proteins in an in vitro system reconstituted from purified components; (3) tag NHEJ components in living cells, induce DSBs in a controlled and synchronous manner, and (4) apply super-sensitive optical imaging methods to analyze the size, composition, and kinetics of assembly and disassembly of the NHEJ machine. We will complement these live-cell imaging studies with cryo-electron microscopy in fixed cells, using dual contrast probes and novel sample preparation methods. We will quantify how fast a nucleoprotein machine runs (kinetics), how accurately it works (accuracy/sensitivity), how quickly it changes its form/shape for different tasks (robustness), and how well it is controlled (feedback/control). The tools, methodologies and results obtained in our short-term and long-term studies of DNA repair machines can be generalized to the studies of other biological systems, including RNA synthesis and processing machines.
To achieve these goals, and to visualize the assembly and disassembly of single NHEJ complexes in the nuclei of living cells, during the first 9 months of our NDC, the biologists, bioengineers, chemists, and computational scientists in the center made a collaborative and concerted effort to develop: (1) small, bright, stable, and biocompatible fluorescent probes; (2) orthogonal tagging strategies to specifically 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. We have made a significant progress over the last 9 months on all these projects and will continue to do so during year-2. Our NDC has attracted nine postdoctoral fellows and two graduate students with a very diverse background, including molecular and cell biology, structural biology, biochemistry, bioengineering, and computational modeling. We plan to create a specialized training program in nanomedicine during year-2 of our NDC as a result of the research and educational activities in the center.
Nearly all human diseases have a genetic component: cancer reflects age-dependent acquisition of somatic mutations, cardiovascular diseases and diabetes reflect inherited metabolic traits, and hemoglobinopathies, lysosomal storage diseases, and inborn errors of metabolism reflect point mutations. 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 NIH Roadmap Initiative in Nanomedicine, we anticipate that, in the future, the allopathic model can be replaced by therapies that directly modify the information contained in DNA and RNA. Therefore, over the last 9 months, we have had extensive discussions to identify the pathway to medicine of our NDC, with a particular focus on the possibility of modifying DNA repair machines to insert, delete, or replace genomic DNA for therapeutic benefits.
We have conceptualized our PtM approach in providing cure for human diseases in terms of three elements: device, delivery, and a specific disease target; we have candidates for each. For the device, we will build on a seminal discovery of the Roth laboratory that the recombination activator gene (Rag) proteins, which normally initiate a specialized V(D)J recombination reaction in developing lymphocytes, can be reengineered to promote site-specific homologous recombination. In principle, this can be achieved for any gene in any cell. For the delivery system, we will adapt an approach based on folate receptor (FR) mediated endocytosis. Folate receptor is commonly overexpressed in cancer cells and there is already a solid foundation of knowledge regarding the use of folate to promote protein and nanoparticle delivery. For the disease, we will address non-small cell lung cancer (NSCLC), which is the most common cause of cancer death worldwide. At the relatively late stage when most diagnosis occurs, there is no effective cure. Any improvement in current therapies would have significant clinical impact. To ensure the clinical relevance and a good chance of success, we will develop close collaborations with clinicians in NSCLC research and, to enhance our effort in PtM, we will recruit two senior faculty members in cancer research to Georgia Tech and Medical College of Georgia as new members of our NDC. Our focus on nucleoprotein machines complements the work of other NDCs. Fundamentally, complex biological functions in living cells are accomplished with only four types of elementary molecular systems: (1) filaments and their networks (the cytoskeleton) that control cell shape and motion, (2) membranes that maintain chemical separation; (3) enzymes that catalyze chemical reactions; and (4) polynucleotides that store and transmit genetic information. Our NDC focuses on nucleoprotein machines that synthesize, modify, or repair DNA and RNA. This complements well with the other NDCs that focus on filaments, membranes, protein enzymes, and photoactivatable biological processes. We have had extensive discussions with other NDCs to develop collaborations and to share resources and expertise.