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. They carry out essential processes of DNA replication, RNA synthesis, pre-mRNA processing, early ribosome biogenesis, RNA transport and DNA damage repair. They can be exceedingly complex – the synthesis and processing of a pre-mRNA, for example, requires precise interaction of hundreds of other RNAs and proteins. Characterization of these machines can be daunting. They assemble transiently, lack a fixed composition, and are often too unstable to isolate for physical analysis. Therefore, significant nanomedicine challenges exist, including:

• To establish the structure-function relationships of each nucleoprotein machine, especially how the machine assemble and disassemble
• To establish signalling and control mechanisms within and between nucleoprotein machines
• To identify engineering design principles shared between nucleoprotein machines of different types

Significant technology development challenges exist in characterizing the dynamics and structure-function 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. It is essential to develop innovative technologies to measure the dynamic behavior of single nucleoprotein machines in living cells to obtain a quantitative description of nanomachines in engineering terms.

Our short-term goal is to develop technologies to visualize the assembly, function, and disassembly of individual nucleoprotein machines in their natural milieu. The long-term goal is to identify the engineering design principles of nucleoprotein machines, and to precisely modify the information stored in DNA and RNA at the single-molecule level, thus providing genetic cures for common human diseases.

Technology Development.
Visualization of single events in the nuclei of living cells is far beyond the limits of existing technology. A collaboration of biologists, bioengineers, chemists, and computational scientists is required to overcome significant technological challenges relating to this goal. Specifically, we will develop:
•Small (< 5 nm), bright, stable, and biocompatible fluorescent probes
•Orthogonal tagging strategies to attach these probes to individual components of a nucleoprotein machine
•Super-resolution imaging technologies to resolve closely spaced probes (20-50 nm) in living cells • Tools to simulate nanomachine formation, to interpret and quantify the experimental data, and from this data and simulation to identify engineering properties of the nanomachines

The Model System.
Initially we will focus on the machine that repairs DNA double strand breaks (DSB) via the nonhomologous end joining (NHEJ) pathway. Reasons for choosing NHEJ include:
•The NHEJ machine is simple compared with others – it has fewer than 10 core components
•NHEJ occurs within self-organizing structures, or “foci”, that are amenable to live-cell visualization
•Assembly of the NHEJ machine can be induced by a single event – one DSB at a defined genomic site
•NHEJ is relevant to the 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

Approaches.
To characterize the NHEJ machine, we willl tag 4-6 of its components using the probes and orthogonal tagging strategies developed by the center. We will validate the activity of the tagged proteins in an in vitro system reconstituted from purified components. We will then tag NHEJ components in living cells, induce DSBs in a controlled and synchronous manner, and apply super-resolution 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.

The design of nucleoprotein machines in living cells has been optimized by nature over billions of years. These machines can realize their functions with astonishing precision, efficiency, and robustness. Successful completion of the goals of our Nanomedicine Development Center will provide a foundation for approaching the truly long-term goal of selective manipulation of DNA and RNA sequence on the nano-scale. We envision that, if this nanomedicine endeavor is successful, we will be able to: (1) engineer nanomachines to physically and topologically isolate their DNA substrate; (2) design different nanomachines that have interchangeable parts, for example, small protein assemblies that execute the same preset series of actions; (3) utilize repeating polymeric structures of DNA and RNA to form natural biological amplifiers, to allow a signal initiating at a single site propagate linearly via chromatin modification, providing a long dock for signaling proteins that arrive, undergo modification, and depart to propagate the signal three-dimensionally. Nearly all human diseases have a genetic component: cancer reflects age-dependent acquisition of somatic mutations, cardiovascular disease and diabetes risk 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 Nanomedicine Roadmap Initiative, we anticipate that, in the future, the allopathic model can be replaced by therapies that directly modify the information contained in DNA and RNA. Our focus on nucleoprotein machines complements the work of other Nanomedicine Development Centers. 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 the other NDCs that focus on filaments, membranes and protein enzymes. We have formed a consortium of nine investigators at eight institutions worldwide. The goals, approaches, and collaborations of our NDC are distinct and higher risk from the research we are pursuing under other support. Although there have been prior collaborations between the imaging and bioengineering laboratories, the involvement of the DNA repair biologists is new, as is the central theme of visualizing single nucleoprotein machines in living cells. Therefore, our NDC fosters new collaborations and develops new approaches.

KEY COLLABORATORS
The composition of the NDC team is as follows:
Name Degree(s) Institutional Affiliation Area of Expertise

Bao, Gang Ph.D. Georgia Institute of Technology, Biomolecular engineering & bionanotech
Dynan, William Ph.D. Medical College of Georgia, Cell & molecular biology; DNA repair
Eils, Roland Ph.D. German Cancer Research Center, Biological modelling and computing
Flores-Rozas, Hernan Ph.D. Medical College of Georgia, Biochemistry; DNA repair
Jensen, Grant Ph.D. California Institute of Technology, Structural biology & cryo-EM
Roth, David M.D./Ph.D. New York University, Molecular biology; V(D)J recombination
Nie, Shuming Ph.D. Emory University, Biomolecular engineering & nanomedicine
Spector, David Ph.D. Cold Spring Harbor Laboratory, Cell & molecular biology; nuclear structure
Ting, Alice Ph.D. MIT, Biochemistry & protein tagging/targeting