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Branford, CT, United States

Disclosed herein is an efficient method of generating a library of variants of a sequence of interest, such as may be used in directed evolution, in one embodiment, the method includes an amplification reaction, e.g. error-prone PCR, to generate double-stranded DNA (dsDNA) variants of a sequence of interest, after which one strand of the dsDNA variants may be selectively degraded to produce single-stranded DNA (ssDNA) variants. The ssDNA variants may be hybridized to ssDNA intermediaries, e.g., uracilated circular ssDNA intermediaries, to form heteroduplex DNA, which may be transformed into cells, such as


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 222.50K | Year: 2014

DESCRIPTION (provided by applicant): The overarching goal of Phase I is to produce a method that will generate, identify and clone a catalog of protein-protein interactions in a single rapid experiment. In this proposal we will model the technology using antibody-antigen interactions. Recombinant antibody libraries against a proteome could be produced prior to any need, and then desired affinity reagents systematically cloned as needed. Libraries of recombinational biopanning integration product against a proteome could also be distributed along with primer sets against a set of barcodes for facile cloning. In future endeavors, the method will be used to clone tumor-cell or other diseased tissue cDNAs to use as an epitope library to quickly identify potential biomarkers. Successful completion of this phase will allow th commercialization of antibodies that can be used for diagnostic and therapeutic applications at a speed and comprehensiveness that is not easily achieved by current methods. Kits composed


Grant
Agency: Department of Health and Human Services | Branch: | Program: STTR | Phase: Phase I | Award Amount: 218.69K | Year: 2014

DESCRIPTION (provided by applicant): Site-specific binding of proteins to DNA plays an important role in cell development, cell signaling, the cell cycle, and diseases such as cancer. There are two overarching goals to this proposal: (1) to develop a meansfor the identification of proteins bound to specific DNA sequences, and (2) to develop a better method for the identification of immunoprecipitation (IP) functional affinity reagents against DNA-bound proteins found on a complex antigen source. We proposeto apply the proof-of-principle using the interaction of transcription factors (TFs) and their specific DNA binding sites as a model. TFs are one major example of such DNA-binding proteins. Once bound to their binding site(s), TF proteins can regulate thetranscription of genes, thereby making individual genes either more or less active. Although this is a proposal to develop a better means toward obtaining functional affinity reagents against proteins when they are bound to DNA, the method itself can


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 334.66K | Year: 2013

DESCRIPTION: We intend to refine the affinity-reagent discovery process so that it is not only cheaper and faster than currently possible, but also so that we produce reagents that are more versatile and useful than current monoclonal antibodies. We propose to use in vitro combinatorial recombination of pre-defined synthetic complementarity determining regions (CDRs) of single chain variable fragment (scFv) antibodies to synthesize a novel type of highly diverse synthetic display library. We have termed this new library-type as a 'pre-defined CDR' (PDC) library. This novel library system will have properties that will also enable it to greatly facilitate both the downstream affinity-reagent selection and the maturation processes. Methods for testing this library using microfluidics and emulsion screening are discussed. PUBLIC HEALTH RELEVANCE PUBLIC HEALTH RELEVANCE: We intend to refine the affinity-reagent discovery process so that it is not only cheaper and faster than currently possible, but also so that we produce reagents that are more versatile and useful than current monoclonal antibodies. A novel library system is proposed that will have properties that will enable it to greatly facilitate both the downstream affinity-reagent selection and the maturation processes. Methods for testing this library using microfluidics and emulsion screening are discussed. This library and the method proposed recapitulates in vitro the way antibodies in vivo are enriched as a response to an antigen assault. Butwe will be able to achieve a comparable analysis on the much larger recombinant libraries (for example 109-1012) we can generate in vitro. Completion of this method has the potential to save time and money and result in a better product over current methods for recombinant affinity-reagent generation.


Grant
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: STTR | Phase: Phase I | Award Amount: 220.32K | Year: 2015

DESCRIPTION provided by applicant Although tyrosine phosphorylation is an important controlling element in cell signaling no tRNA suppressor for phosphotyrosine pTyr incorporation has yet been made We propose to use directed molecular evolution of several aminoacyl tRNA synthetases aaRSs to identify mutations that enable binding of pTyr to the aaRS Specifically ATP molecules will be attached to beads which will then be incubated with free pTyr and a phage display library of a mutated aaRS If a mutant aaRS can catalyze the formation of pTyr AMP which is the intermediate for the generation of charged tRNA it will bind to the beads and can be enriched The mutated aaRSs will be used in in vitro translation to incorporate the pTyr into the protein structure of an assayable gene for example galactosidase Mass Spectrometry and a set of already existing anti pTyr specific antibodies will be used to validate incorporation of the pTyr in the assayable protein Phase I is focused on in vitro incorporation PUBLIC HEALTH RELEVANCE The ability to generate tyrosyl phosphorylated proteins will have significant utility in studying the role of phosphoproteins that are involved in cell signalin inflammation cancer and other diseases Conventional approaches toward making phosphorylated proteins require kinases which are promiscuous and often lead to phosphorylation at multiple undesirable residues Successful completion of this proposal will be the first method to allow site specific incorporation of phosphotyrosine in a protein

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