Nanomedicine Center For Nucleoprotein Machines

Nucleoprotein Machines

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.

Nanomedicine Development Center Goal

Our goal is to develop technologies to visualize the assembly, function, and disassembly of individual nucleoprotein machines in their natural milieu. Many biochemical and cell biological methods measure the average behavior of ensembles of molecules, but such approaches are inherently limited because they rely on an untested and implausible assumption that each nucleoprotein machine of a given class is the same. To transcend this limitation, it is essential to be able to observe and analyze single nucleoprotein machines. Only by achieving this goal will we be able to obtain a quantitative description of the composition and behavior of nucleoprotein machines in engineering terms. We hope to identify common principles that can be applied to the design of artificial nucleoprotein machines with novel specificities, facilitating the precise manipulation of DNA and RNA at the atomic level.

Technology Development

An Example Of A Simple Nucleoprotein Machine

To characterize the NHEJ machine, we willl tag 4-6 components using the probes and 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 insert the tagged proteins into 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).

Nanomedicine: Common Versus Distinct Themes

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 atomic 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 values and spirit 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 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.

Investigative Team

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. The composition of the team is as follows: