We will use our computational design methodology, based on an explicit physical model of protein-DNAinterfaces, to design novel homing endonuclease variants predicted to cleave specifically within sites inXSCID and other therapeutically important genes. Genes corresponding to the designed proteins will besynthesized, the in vitro and in vivo cleavage specificities determined in Component 2 - Monnat, Component4 - Scharenberg, and Component 5 - Stoddard groups, improved variants obtained using molecularevolution 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 ofbackbone flexibility and of water-mediated hydrogen bonding interactions which can contribute significantlyto the energetics of protein-DNA interactions, and we will use the experimental feedback to improve bothaspects of our model; for example the crystal structures will guide our approach to modeling backboneflexibility. We will use the improved computational design methods to design a second round ofendonucleases with therapeutically important cleavage specificities and these will be characterized as in thefirst round and the experimental feedback used to further refine the computational method. By cycling in thisway between detailed computational modeling and in depth experimental characterization we aim to developrobust methods for creating tailored enzymatic reagents for targeted genetic therapies via gene correction.
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