Nucleoprotein machines carry out essential biological processes including synthesis, modification, and repair of DNA and RNA. We propose to establish a nanomedicine development center (NDC) focusing on a model nanomachine that carries out nonhomologous end joining (NHEJ) of DNA double strand breaks. This and other DNA repair machines have relatively simple structures (<20 components) and significant biological and clinical relevance. 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 have assembled an interdisciplinary team from eight institutions, with significant expertise in cell and molecular biology of DNA damage repair, protein tagging and targeting, nanostructured probes, cryo-electron microscopy, signal-cell imaging, quantitative image analysis and computational biology, and light microscopy instrumentation. We will develop innovative nanotechnologies and biomolecular approaches to elucidate the structure-function relationships within and among DNA repair nanomachines. General principles emerging from these studies will lay a foundation for precise modification of the information stored in DNA and RNA, leading ultimately to novel therapeutic strategies for a wide range of diseases, including cancer. The NDC has five closely related aims including: (1) to develop orthogonal protein tagging strategies and novel fluorescence probes including quantum dot bioconjugates for nanomachine targeting;(2) to decipher structure-function relationship of components required for the core NHEJ reaction;(3) to characterize the dynamics of nanomachine assembly and disassembly in the context of repair foci;(4) to determine the dimensions and structure of repair foci at high resolution in fixed cells;(5) to establish the engineering design principles underlying DNA double-strand break repair. This NDC will complement existing NDCs that focus on filaments, membranes and protein folding enzymes, and the probes, tools and methodologies developed will be applicable to a wide range of biological and disease studies. 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 approaches that are inexpensive, effective, and user-friendly, similar to vaccination today.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Development Center (PN2)
Project #
5PN2EY018244-10
Application #
8725160
Study Section
Special Emphasis Panel (ZEY1-VSN (20))
Program Officer
Fisher, Richard S
Project Start
2006-09-30
Project End
2015-07-31
Budget Start
2014-08-01
Budget End
2015-07-31
Support Year
10
Fiscal Year
2014
Total Cost
$3,200,000
Indirect Cost
$348,531
Name
Georgia Institute of Technology
Department
Engineering (All Types)
Type
Schools of Engineering
DUNS #
097394084
City
Atlanta
State
GA
Country
United States
Zip Code
30332
Lin, Yanni; Cradick, Thomas J; Bao, Gang (2014) Designing and testing the activities of TAL effector nucleases. Methods Mol Biol 1114:203-19
Voit, Richard A; Hendel, Ayal; Pruett-Miller, Shondra M et al. (2014) Nuclease-mediated gene editing by homologous recombination of the human globin locus. Nucleic Acids Res 42:1365-78
Lin, Yanni; Cradick, Thomas J; Brown, Matthew T et al. (2014) CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences. Nucleic Acids Res 42:7473-85
Hendel, Ayal; Kildebeck, Eric J; Fine, Eli J et al. (2014) Quantifying genome-editing outcomes at endogenous loci with SMRT sequencing. Cell Rep 7:293-305
Lin, Yanni; Fine, Eli J; Zheng, Zhilan et al. (2014) SAPTA: a new design tool for improving TALE nuclease activity. Nucleic Acids Res 42:e47
Fine, Eli J; Cradick, Thomas J; Zhao, Charles L et al. (2014) An online bioinformatics tool predicts zinc finger and TALE nuclease off-target cleavage. Nucleic Acids Res 42:e42
Qiu, Yongzhi; Brown, Ashley C; Myers, David R et al. (2014) Platelet mechanosensing of substrate stiffness during clot formation mediates adhesion, spreading, and activation. Proc Natl Acad Sci U S A 111:14430-5
Tong, Sheng; Fine, Eli J; Lin, Yanni et al. (2014) Nanomedicine: tiny particles and machines give huge gains. Ann Biomed Eng 42:243-59
Lindsay, Cory R; Roth, David B (2014) An unbiased method for detection of genome-wide off-target effects in cell lines treated with zinc finger nucleases. Methods Mol Biol 1114:353-69
Fine, Eli J; Cradick, Thomas J; Bao, Gang (2014) Identification of off-target cleavage sites of zinc finger nucleases and TAL effector nucleases using predictive models. Methods Mol Biol 1114:371-83

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