Nonhomologous Random Recombination of Nucleic Acids (Nucleic Acid Shuffling)

Summary This invention comprises methods for non-homologous random recombination (NRR) of nucleic acids, and their use to develop and select nucleic acids or polypeptides with novel structure and function. This technology is distinct from previous DNA shuffling methods in that it provides more comprehensive recombination of nucleic acid sequences for non-natural evolution.

Background:

In vitro DNA mutagenesis and shuffling enable the non-natural "molecular evolution" of nucleic acid sequences and the proteins they encode. This allows selected protein characteristics to be manipulated to improve their commercial value. Molecular evolution is readily applied to proteins engineered by recombinant DNA technology, which have already demonstrated the economic importance of non-natural proteins. The fields of pharmaceuticals, agriculture, and chemistry have shown billion dollar annual sales of non-natural protein products, many of which could be improved through molecular evolution. Enzymes currently in use as thermostable detergents and as PCR reagents were developed using molecular evolution. In addition to the protein products they encode, nucleic acid libraries created by the method can be used to create catalytic RNA or DNA, or to make DNA aptamers that could replace proteins in molecular recognition events. Successful molecular evolution requires the efficient exploration of sequence space to locate molecules encoding the desired properties. Previous methods of introducing diversity include whole-genome mutagenesis, error-prone PCR, random cassette mutagenesis, and DNA shuffling. Of these, only DNA shuffling allows the recombination of parent DNA sequences, but it is still limited to recombination at sites of homology between sequences. Studies have shown that NRR could drive the production of hybrid enzymes with activity greater than those hybrids formed by traditional DNA shuffling.

Applications The NRR method described allows portions of nucleic acids to be recombined at sites where there is little or no sequence homology. This will increase the speed at which novel sequences are generated, allowing more rapid evolution of nucleic acid aptamers, DNA and RNA catalysts, and proteins with unique folding and function. For Further Information Please Contact the Director of Business Development Vivian Berlin Email: [email protected] Telephone: (617) 495-0474

Inventor(s): Liu, David R

Type of Offer: Licensing



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