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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 natural machines that repair DNA double-strand breaks (DSBs), including the non-homologous end joining (NHEJ) and homologous recombination (HR) complexes. These two competing pathways of DSB repair, NHEJ and HR, occur within self-organizing structures, or “repair foci” that are amenable to live-cell visualization using technologies developed in the NDC. The short-term goal of this NDC is to characterize the assembly of DSB repair complexes using molecular imaging approaches. We will characterize the assembly of nucleoprotein machines in vitro, using purified proteins, and in vivo, within living cells. DSB repair is one of a few natural processes that permit a cell to alter its own genetic content, suggesting the possibility that repair pathways might be redirected for gene editing and gene correction. Consistent with the vision of the NIH Nanomedicine Initiative, the long-term goals of our NDC are bold and ambitious: to obtain a general understanding of engineering design principles of DNA repair machines, and to use this understanding to develop a means to precisely modify the information stored in DNA, thus providing novel therapeutic strategies for a wide range of human diseases. Sickle cell disease, a devastating, common, and incurable hemoglobinopathy, will be the focus of our Pathway to Medicine (PtM). We aim to treat sickle cell disease by using nucleases and nucleoprotein machines to correct the A-T mutation of the beta-globin gene.
The mammalian cell nucleus is filled with self-organizing, interconnected, nanometer-scale machines that carry out numerous fundamental processes such as DNA replication, RNA synthesis, RNA transport and DNA repair. We refer to a general class of these nanomachines that are made primarily of protein and act on nucleic acid substrates as “nucleoprotein machines.” They are complex: synthesis of a typical human mRNA, for example, requires interaction of hundreds of protein and RNA components, including initiation, capping, elongation, splicing, polyadenylation and termination factors.
We aim to establish the structure-function relationship of specific nucleoprotein machines, including how the machines assemble and disassemble and the signalling and control mechanisms. Nucleoprotein machines work with a common set of raw materials (nucleotides and polynucleotides), carry out similar elementary steps (nucleotidyl and phosphoryl group transfer), and often have interchangeable components. These similarities suggest that the study of different nucleoprotein machines will reveal common, and generalizable engineering design principles. Nucleoprotein machines often do not have a fixed composition. They are dynamic with proteins associating and dissociating depending on the specific functional state of the biological process. For example, we study the nonhomologous end joining (NHEJ) and homologous recombination (HR) complexes that repair DNA double strand break (DSB) in mammals. NHEJ and HR occur within chromatin domains of roughly 2 million base pairs of DNA and must be able to join ends of virtually any nucleotide sequence. Since each repair complex is unique at any instant of time, it is essential to study the behavior of single nucleoprotein complexes, rather than population averages, in order to gain insight into their design and function.
A long-term goal of our NDC is to develop a new gene correction approach to precisely modify the information stored in DNA using nucleoprotein machines, thus providing novel therapeutic strategies for a wide range of human diseases. As our pathway to medicine (PtM), we will explore the use of nucleoprotein machines, especially the HR complex, in conjunction with nucleases, to correct the A-T mutation of the beta-globin gene for treating sickle cell disease, a devastating, common, and incurable human disease. If successful, this same approach can be applied to treating other human diseases, such as Huntington’s disease.
Modern medicine – allopathic medicine – focuses on treating symptoms, commonly through small-molecule enzyme inhibitors and receptor agonists/antagonists. It does not address the underlying genetic causes of disease. Consistent with the vision of the NIH Nanomedicine Initiative, our NDC envisions that the allopathic model will someday be replaced by therapies that directly modify information contained in a patient’s DNA.
Why the NHEJ and HR Complexes?
Initially we focus our studies on the nonhomologous end joining (NHEJ) pathway that repairs DNA DSB because:
- The NHEJ complex is simple – it has fewer than 10 core components
- NHEJ occurs within self-organizing structures, “foci”, that are amenable to live-cell visualization
- Assembly of the NHEJ machine can be induced by a single DSB at a defined site on a DNA strand
- NHEJ is already used by the immune system to create novel antigen receptors in V(D)J recombination (see below). Understanding this function will provide clues for us to adapt the complex for manipulating of DNA or RNA.
More recently we have also been working on the homologous recombination (HR) pathway for DSB repair because:
What do we know about how NHEJ and HR fix broken DNA?
- The homologous recombination complex is also relatively simple, with fewer than 10 core components
- HR requires the presence of an identical or nearly identical sequence to be used as a template for repair of DSB
- HR is capable of producing new combinations of DNA sequences
- It is important to study both NHEJ and HR pathways, especially what controls pathway choice (e.g., cell-cycle dependent regulation of HR and NHEJ)
- Understanding similarities and differences in the design principles of NHEJ and HR
A working model of the NHEJ and HR complexes is shown in Figure 1. HR utilizes a homologous stretch on a sister chromatid to accurately repair the DSB. DNA ends are first processed in order to create single-strand overhangs, a process that is likely mediated by the MRN(Mre11/Rad50/ Nbs1) complex. Rad51, Rad52, and RPA associate with these overhangs, followed by the formation of a joint molecule by the damaged and undamaged strands. Template guided DNA synthesis and resolution of the two strands then complete repair of the DSB. To form the NHEJ complex, the Ku protein (composed of Ku70 and Ku80 subunits) carries out initial end recognition. Ku recruits the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), which sequesters the DNA ends. The DNA ligase complex (composed of DNA ligase IV, XRCC4, and XLF) then catalyzes phosphodiester bond formation. These six polypeptides are sufficient for the core NHEJ reaction. Mammalian cells that are deficient in any of the core NHEJ components are very sensitive to DSB-inducing agents. Most of the core NHEJ components (except DNA-PKcs) are required for an analogous NHEJ pathway in budding yeast, which has allowed analysis of NHEJ in this genetically tractable model organism.
To fully understand the design of nucleoprotein machines, it is necessary to characterize the dynamics of the NHEJ and HR complexes in living cells. However, visualizing single NHEJ and HR complexes in the nuclei of living cells is far beyond the limits of existing technology, and progress toward this goal requires a multidisciplinary team of biologists, bioengineers, chemists, and computer scientists.
Development and application of protein tagging strategies and imaging methods.
Our first step is to synthesize new optical imaging probes that are small (4-6 nm) and biocompatible and develop new strategies to attach these probes to individual components of the NHEJ and HR complexes. After labelling 2, 3 or 4 components, we can visualize machine assembly in vitro using single molecule techniques. We have been using optical imaging methods with improved sensitivity and spatial resolution to characterize the assembly and disassembly of the repair nanomachine on a chromatin substrate in living cells. Developing new imaging capabilities also allows us to 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.
Characterization of assembly of HR and NHEJ complexes and pathway choice.
We have been able to induce DSBs in vivo in a controlled manner, using several complementary technical approaches, including laser scanning, microirradiation, and the use of zinc finger nucleases (ZFNs). To characterize the initial steps in assembly of single HR and NHEJ complexes deep inside living cells, we combine the new imaging probes with high-sensitivity, high-resolution optical imaging instrumentation. In parallel studies, we have been developing an in vitro single-molecule detection system to track repair complex assembly based on the creation of a site-specific DSB within a topologicaly isolated loop. 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.
When DSBs are generated, in most of the cases (~99%) they are repaired by the NHEJ. However, for correcting genetic defects by creating a DSB on the target DNA, it is necessary to trigger the HR pathway. Therefore, studies are being performed to understand pathway choices in repairing DSB, i.e., how cells choose between NHEJ and HR pathways. We are also exploring ways to alter pathway choice by, for example, selectively blocking the NHEJ pathway and/or encouraging the HR pathway.
Establishment of new gene correction approaches for treating sickle cell disease.
The gene correction machines are based on ZFN or RAG-mediated DNA recognition and cleavage; they will be used initially to correct the A-T mutation in sickle β-globin gene as a novel method to treat sickle cell disease (SCD). Major efforts in our NDC includes: (1) development of ZFNs that specifically create a single DSB in a β-globin gene adjacent to the sickle mutation, (2) demonstration of targeted delivery of nucleases and template to the nuclei of living cells, including the hematopoietic stem/progenitor cells (HSPCs) that will be the ultimate target for gene correction, (3) demonstration that RAG proteins can induce site-specific nick with a high degree of specificity, and promote efficient gene conversion, and (4) as a significant step toward the development of a clinically viable (safe and efficacious) method, to demonstrate the efficacy of gene correction based approach in treating sickle cell disease using an animal model of SCD.
In summary, as shown in Fig. 2, this NDC has three major thrust areas: (1) new methodology development, including developing protein tagging strategies and imaging probes, in vitro assays of NHEJ/HR, live-cell imaging methods, and new modeling and image analysis approaches; (2) basic biological studies of NHEJ/HR assembly/disassembly, including structure-function relations of NHEJ/HR, pathway choice, and methods to alter pathway choice; (3) as a pathway to medicine, establishing a novel strategy to treat sickle cell disease, including the generation of site-specific ZFNs/RAGs for ‘cutting’, in vivo delivery of ZFNs/RAGs and the template, and demonstration of the gene correction efficacy using an animal model of SCD. These three thrust areas in research and education are closely related and fully integrated, and efforts will be made to advance both basic and translation research.
The five (5) most important scientific advances by our NDC since our NDC began are given below:
Fundamentally orthogonal labeling methods.
Orthogonal in vivo labeling of NHEJ and HR is a main goal of this NDC. We labeled Ku80, XRCC4, 53BP1 in live cells with SNAP, ReAsh, and HaLo. Labeling was performed in situ in the nuclei of living cells. The tagged proteins showed clear nuclear localization ands were recruited to DSBs. A fourth labeling method, based on an evolved coumarin ligase and 13 amino acid acceptor peptide (LAP2) has been demonstrated in living cells and we are currently adapting it to the DSB response proteins, 53BP1 and RAD52. We are also working to demonstrate various combinations of the fundamentally orthogonal methods simultaneously in the same cell.
Compact and strain-tuned QDs.
We developed ultrasmall biocompatible QDs with novel polymer coatings for intracellular protein tagging. We also developed strain-tuned quantum dots (QDs) that show narrow light emission spectra with high quantum yields (60%), and are tunable across a broad range of visible and near-infrared wavelengths (500 nm to 1050 nm). The best of our current QDs are only 5.6-9.7 nm with coating, reducing their bulk (inversely proportional to radius cubed) up to 50-fold relative to the smallest biocompatible commercial QDs.
A critical issue in using QDs to tag NHEJ/HR components is the size: the conventional QDs are ~20 nm in size, thus are likely to interfere with assembly. We have developed a new strategy to minimize the hydrodynamic size of QDs based on the use of multifunctional and multidentate polymer ligands. A major finding is that a mixed composition of thiol (-SH) and amine (-NH2) coordinating groups grafted to a linear polymer chain can lead to a highly compact QD with long-term colloidal stability, a strong resistance to photobleaching, and high fluorescence quantum yield. In contrast to the standing brush-like conformations of PEGylated dihydrolipoic acid ligands and monovalent thiols, we believe that these multidentate polymer ligands can wrap around the QD in a closed “flat” conformation. This structure is highly stable from a thermodynamic perspective, and is thus responsible for the excellent colloidal and optical properties observed. As a result, we have prepared a new generation of bright and stable QDs with small hydrodynamic sizes between 5.6 nm to 9.7 nm, with fluorescence emission tunable from the visible (515 nm) to the near infrared (720 nm). Figure 2 shows a size comparison of gel filtration chromatograms of multidentate polymer-coated quantum dots (four emission colors) with globular protein standards. The results demonstrate that the green-emitting QDs (515 nm) have a hydrodynamic size slightly larger than fluorescent proteins (MW = 27-30 kDa), while the yellow-emitting quantum dots (562 nm) dots are slightly smaller than serum albumin (MW = 66 kDa). Even the near-infrared emitting dots (720 nm) are similar to antibodies (MW = 150 kDa) in hydrodynamic size.
Fluorogenic protein beacons.
We developed small, orthogonal, fluorogenic protein beacons with the potential to target endogenous proteins in live cells. The design of the protein beacons has been optimized, and preliminary cellular studies have shown labeling specificity. To our knowledge this system may provide the only general method for studying the interaction of endogenous proteins in living cells.
Visualization of recruitment of NHEJ/HR at ZFN induced DSBs.
We visualized co-recruitment of 53BP1 (tudor domain) Ku80, and XRCC4 to a transgene array in human U2OS 2-6-3 cells. The 53BP1 signal was far higher than for Ku80 and XRCC4, consistent with the assumption that 50-100 copies of 53BP1 are present at each DSB, but only 2 copies of Ku70/80 and XRCC4. To our knowledge this will be the first reported observation of recruitment of core NHEJ components (Ku and XRCC4) to a defined DSB.
Our most exciting results to date have resulted from combining two different tools, which were developed independently in two laboratories of the NDC. One is a human cell line containing a transgene array, composed of interspersed lac operator and ECFP coding sequences. This is stably integrated into the genome of human U2OS cells at 1p36. Electroporation of a LacI-fluorescent fusion protein expression construct into these cells results in binding of the LacI-fusion protein to the transgene locus, which appears as a single fluorescent spot in the nuclear interior. The other tool is a pair of chimeric zing finger nucleases (ZFN1 and ZFN2), developed by the Porteus Laboratory, that recognize a sequence within the integrated ECFP gene. The ZFNs combine an engineered zinc finger domain (which can be designed to recognize any arbitrarily preselected DNA sequence) with the non-specific cleavage domain of the Fok I restriction exonuclease.
Targeted inhibition of NHEJ function in living cells.
We developed a method, based on folate receptor mediated endocytosis, that allows efficient delivery of an engineered antibody fragment to the cell nucleus. The antibody binds a specific site in the NHEJ complex and blocks regulatory autophosphorylation, resulting in quantifiable radiosensitization. To our knowledge this will be the first method (other than microinjection or transgenesis) for delivery of a protein therapeutic to the mammalian cell nucleus).
Pathway to Medicine
A long-term goal of our NDC is to adapt DNA repair nanomachines for therapeutic benefits by precisely modifying the information stored in DNA and RNA, thus providing genetic cures for common human diseases. 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 NIH Nanomedicine Initiative, we anticipate that, in the future, the allopathic model can be replaced by therapies that directly modify information contained in DNA or RNA.
We elected to focus our Pathway to Medicine (PtM) efforts on treating sickle cell disease (SCD), which is caused by a single-gene (b-globin) defect (A-T mutation). Even modestly efficient gene correction, by reducing the fraction of sickle hemoglobin, will result in improved patient outcome. Further, SCD is life shortening, painful, reasonably common, and contributes to minority health disparities (afflicting primarily persons of African origin in the US). Importantly, the only curative therapy (bone marrow transplantation) is practical in only a small subset of patients. Our approach is to use engineered nucleases (zinc finger nucleases (ZFNs, see Fig. 4) or modified RAG nucleases) to generate a DNA double strand break (or a nick) near the mutation site in progenitor cells of the patient, which activates the HR pathway and fixes the mutation. Our ten-year goal is to establish this novel gene correction method and to demonstrate its efficacy in correcting the mutation of the b-globin gene in a sickle-cell transgenic mouse model. Specifically, our Pathway to Medicine effort has the following major components:
- To understand pathway choices. When a DSB or nick is generated, how cells choose between NHEJ and HR pathways and how to construct a nucleoprotein gene correction machine that selectively engages the HR pathway for gene correction at a target locus.
- To develop clinically viable methods for delivery of a nucleoprotein machine to the relevant cell type. Initially, we will work with an established human erythroleukemia cell line, but in parallel, we will develop methods for delivery to isolated human murine hematopoietic stem cells (HSC), which can be reimplanted following gene correction.
- To determine the efficiency of engraftment of HSC in a recipient mouse and quantify reversal of the disease phenotype.
- Upon success in pre-clinical studies, to carry out phase 1 clinical trials with sickle cell disease patients through a collaboration with clinicians.
Although the above PtM efforts may take many years, we envision that, through the research efforts in our NDC over the next few years (2-5 years), we will have a much better understanding of how to perform gene correction precisely, safely and efficiently using nucleoprotein machines. If our efforts are successful, we will be able to demonstrate the feasibility of treating sickle cell disease using the novel gene correction approach and open a new avenue of curing other human diseases.
Current NDC supported papers (9/30/06-9/29/09)
- Dynan WS, Li S, Takeda Y. Modifying the Function of DNA Repair Nanomachines for Therapeutic Benefit. Nanomedicine: Nanotechnology, Biology, and Medicine, 2, 74-81 (2006).
- Ruan G, Agrawal, Marcus AI and Nie SM. Imaging and Tracking Tat-Peptide Conjugated Quantum Dots in Living Cells: New Insights into Nanoparticle Uptake, Intracellular Transport, and Vesicle Shedding. J Am Chem Soc, 129, 14759-14766 (2007). PMID: PMC17983227
- Corneo B, Wendland RL, Deriano L, Cui X, Klein IA, Wong SY, Arnal S, Holub AJ, Weller GR, Pancake BA, Shah S, Brandt VL, Meek K, Roth DB. Rag mutations reveal robust alternative end joining. Nature, 449, 483-486 (2007). PMID: PMC17898768
- Smith AM, Duan H, Mohs A and Nie SM. Bioconjugated quantum dots for in-vivo molecular and cellular imaging. Adv. Drug Delivery Rev, 60, 1226-40 (2008). PMID: PMC18495291
- Smith AM and Nie SM. Minimizing the Hydrodynamic Size of Quantum Dots with Multifunctional Multidentate Polymer Ligands, J. Am. Chem. Soc., 130, 11278-11279 (2008). PMCID: PMC18680294
- Kairdolf BA, Smith AM, and Nie SM. One-Pot Synthesis, Encapsulation, and Solubilization of Size-Tuned Quantum Dots with Amphiphilic Multidentate Ligands, J. Am. Chem. Soc., 130, 12866- 12867 (2008). PMCID: PMC18774812
- Dynan WS, Takeda Y, Roth DB, and Bao G. Understanding nucleoprotein machines and reengineering them to cure human diseases. Nanomedicine, 3, 93-105 (2008). NIHMSID #129473
- Dimitrova N, Chen Y-CM, Spector DL and De Lange T. 53BP1 promotes non-homologous end joining of telomeres by increasing chromatin mobility. Nature 456, 524-528 (2008). PMCID: PMC2613650
- Nitin N, LaConte L, Rhee W J and Bao G. 2008. Tat Peptide Is Capable of Importing Large Nanoparticles across Nuclear Membrane. Ann. Biomed. Eng., 2009 Aug 6. [Epub ahead of print]. NIHMSID #137549
- Smith AM, Mohs AM, and Nie SM. Tuning the optical and electronic properties of colloidal nanocrystals by lattice strain, Nature Nanotechnology 4, 56-63 (2009). PMCID: PMC19119284
- Smith AM, Wen MM, Wang MD, and Nie SM. 2009. Size-Minimized Quantum Dots for Molecular and Cellular Imaging, in Novel Symposium on Single-Molecule Chemistry, Physics, and Biology, Stockholm, Sweden. NIHMSID# 129719
- Xiong H, Li S, Yang Z, Burgess RR, and Dynan WS. E. coli expression of soluble, active scFv variants containing a nuclear localization signal. Protein Expr. Purif. 66, 172-180 (2009). Pending PMCID
- Li S, Nguyen L, Xiong H, Wang M, Hu T C-C, She J-X, Serkiz SM, Wicks GG, and Dynan WS. Porous-wall hollow glass microspheres as novel carriers for biomolecules. Nanomedicine, Jul 15 2009, Epub ahead of print. Pending PMCID
- Bao G. Protein Mechanics, Experimental Mechanics 49, 153-164 (2009). NIHMSID #116450
- Bao G, Santangelo P, Nitin N, and Rhee WJ. Nanostructured Probes for In Vivo Gene Detection. In Nanomedicine (Ed. Viola Vogel), Nanotechnology Series, WILEY-VCH, Weinheim, Germany (2009). NIHMSID #132479
- Slavoff SA, Chen I, Choi YA, and Ting AY. Expanding the substrate tolerance of biotin ligase through exploration of enzymes from diverse species. , J. Am. Chem. Soc. 130, 1160-1162 (2008). PMID: PMC18171066
- Puthenveetil S, Liu DS, Thompson S, and Ting AY. Yeast display evolution of a kinetically efficient 13-amino acid substrate for lipoic acid ligase. Submitted (2009).
Contact information for the PI and key co-PIs:
|Bao, Gang (PI)
||Georgia Institute of Technology
||Medical College of Georgia
||German Cancer Research Center
||California Institute of Technology
||Medical College of Georgia
||UT Southwestern Medical Center
||New York University
||Cold Spring Harbor Laboratory
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