We will use our computational design methodology, based on an explicit physical model of protein-DNA interfaces, to design novel homing endonuclease variants predicted to cleave specifically within sites in XSCID and other therapeutically important genes. Genes corresponding to the designed proteins will be synthesized, the in vitro and in vivo cleavage specificities determined in Component 2 - Monnat, Component 4 - Scharenberg, and Component 5 - Stoddard groups, improved variants obtained using molecular evolution by Component 4 - Scharenberg, and structures determined of promising designs in Component 5 - Stoddard. These data will be used to refine and improve our computational design methodology. Shortcomings of the physical model underlying our current approach include the limited treatments of backbone flexibility and of water-mediated hydrogen bonding interactions which can contribute significantly to the energetics of protein-DNA interactions, and we will use the experimental feedback to improve both aspects of our model;for example the crystal structures will guide our approach to modeling backbone flexibility. We will use the improved computational design methods to design a second round of endonucleases with therapeutically important cleavage specificities and these will be characterized as in the first round and the experimental feedback used to further refine the computational method. By cycling in this way between detailed computational modeling and in depth experimental characterization we aim to develop robust methods for creating tailored enzymatic reagents for targeted genetic therapies via gene correction.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Linked Research project Grant (RL1)
Project #
5RL1GM084433-04
Application #
7884491
Study Section
Special Emphasis Panel (ZRR1-SRC (99))
Program Officer
Hagan, Ann A
Project Start
2007-09-17
Project End
2012-06-30
Budget Start
2010-07-01
Budget End
2011-06-30
Support Year
4
Fiscal Year
2010
Total Cost
$378,760
Indirect Cost
Name
University of Washington
Department
Biochemistry
Type
Schools of Medicine
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195
Thyme, Summer; Song, Yifan (2016) Computational Design of DNA-Binding Proteins. Methods Mol Biol 1414:265-83
Baxter, Sarah K; Scharenberg, Andrew M; Lambert, Abigail R (2014) Engineering and flow-cytometric analysis of chimeric LAGLIDADG homing endonucleases from homologous I-OnuI-family enzymes. Methods Mol Biol 1123:191-221
Thyme, Summer; Baker, David (2014) Redesigning the specificity of protein-DNA interactions with Rosetta. Methods Mol Biol 1123:265-82
Thyme, Summer B; Boissel, Sandrine J S; Arshiya Quadri, S et al. (2014) Reprogramming homing endonuclease specificity through computational design and directed evolution. Nucleic Acids Res 42:2564-76
Thyme, Summer B; Song, Yifan; Brunette, T J et al. (2014) Massively parallel determination and modeling of endonuclease substrate specificity. Nucleic Acids Res 42:13839-52
Baxter, Sarah; Lambert, Abigail R; Kuhar, Ryan et al. (2012) Engineering domain fusion chimeras from I-OnuI family LAGLIDADG homing endonucleases. Nucleic Acids Res 40:7985-8000
Thyme, Summer B; Baker, David; Bradley, Philip (2012) Improved modeling of side-chain--base interactions and plasticity in protein--DNA interface design. J Mol Biol 419:255-74
Szeto, Mindy D; Boissel, Sandrine J S; Baker, David et al. (2011) Mining endonuclease cleavage determinants in genomic sequence data. J Biol Chem 286:32617-27
Windbichler, Nikolai; Menichelli, Miriam; Papathanos, Philippos Aris et al. (2011) A synthetic homing endonuclease-based gene drive system in the human malaria mosquito. Nature 473:212-5
Zhang, Junjie; Ma, Boxue; DiMaio, Frank et al. (2011) Cryo-EM structure of a group II chaperonin in the prehydrolysis ATP-bound state leading to lid closure. Structure 19:633-9

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