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Modified Nucleotidyl Transferases for Enzymatically Mediated Oligodeoxynucleotide Synthesis

用于酶介导的寡脱氧核苷酸合成的修饰核苷酸转移酶

基本信息

批准号：
8904404
负责人：
John W Efcavitch
金额：
$ 18.9万
依托单位：
MOLECULAR ASSEMBLIES, INC.
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-08-01 至 2016-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8904404
关键词：
Active Sites Aliquot Amino Acids Automation Binding Biological Biological Assay Biology Budgets Buffers Cells Collaborations Computing Methodologies Custom DNA DNA Nucleotidylexotransferase DNA Sequence DNA biosynthesis DNA chemical synthesis Development Engineering Enzymes Escherichia coli Failure Gel Genes Genomics Goals Health Health Services Research Hour Human Ions Laboratories Lead Length Libraries Link Manuals Mediating Methods Modeling Modification Molecular Biology Molecular Models Monitor Mutation N-terminal Natural regeneration Nucleic Acids Nucleotides Oligonucleotides Organic Chemistry Phase Plant Resins Play Polymerase Polynucleotides Positioning Attribute Process Proteins Reaction Reagent Recombinants Role Science Services Single-Stranded DNA Solid Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization Spottings Synthetic Genes Technology Therapeutic Time Transferase Transferase Gene Validation Variant analog aqueous base clinical application combinatorial cost design enzyme activity expression vector fictional works gene synthesis improved model design molecular assembly/self assembly molecular modeling mutant novel nucleotide analog phosphoramidite prototype public health relevance screening synthetic biology synthetic construct tripolyphosphate vaccine development wasting

项目摘要

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 (>500bp) 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 1981 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 & clinical applications.