Modified Nucleotidyl Transferases for Enzymatically Mediated Oligodeoxynucleotide Synthesis

DESCRIPTION provided by applicant In the rapidly growing field of synthetic biology the cost of synthetic DNA for gene synthesis has become a substantial part of many laboratory budgets Current DNA synthesis technologies are unable to produce gene length DNA fragments and rely on expensive and error prone assembly methods to construct long strands of DNA In addition current DNA synthesis methods are organic chemistry based and produce toxic waste mixtures that are difficult and costly to dispose of Here we propose to design a novel biologically based DNA synthesis method which will enable the high fidelity template independent synthesis of long andgt bp strands of DNA This proposal describes a novel method that will lead to reduced costs in every synthetic biology laboratory enabling applications such as faster development of vaccines biomolecular computation reprograming of cells and improved cellular therapeutics The resulting massively parallel synthesis capability developed in Phase II of this project will be put to use as a custom synthesis service by Molecular Assemblies similar to how custom oligos are ordered produced and delivered today To achieve this this proposal focuses on engineering the enzyme terminal deoxynucleotidyl transferase TdT which acts by adding nucleotides to single stranded DNA in a template independent fashion to utilize modified deoxynucleotide triphosphates dNTPs The dNTP analogs are blocked in such a way that leads to the addition of one and only one nucleotide of choice at a time and after being added to the growing strand are able to be de blocked to regenerate a natural DNA strand Phase I covers the development of an engineered enzyme and its use to prove the ability to make a short sequence specific nucleic acid Phase II will lead to optimization of all four dNTP analogs cycle conditions automation and the demonstration of the full capabilities of this novel synthetic approach for polydeoxynucleotides In it would have been difficult to envision the specific fundamental roles DNA synthesis would eventually play in modern biology while the idea of purchasing synthetic genes was a concept of science fiction One can only imagine how on demand high purity low cost polynucleotides will enable a new era of biological andamp clinical applications PUBLIC HEALTH RELEVANCE The cost of synthetic DNA for routine applications such as DNA sequencing PCR and hybridization analysis has become a substantial part of many laboratory budgets Additionally length and quality issues limit the expansion of numerous biological applications which could impact human health developments This proposal describes a method that will reduce costs in every molecular biology laboratory and will enable the development of new synthetic DNA applications such as faster development of vaccines biomolecular computation reprograming of cells and improved cellular therapeutics