Compositions and methods related to nucleic acid synthesis
Inventors
Chen, Michael C. • Lazar, Radu A. • Huang, Jiahao • McInroy, Gordon R.
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Assignees
Member
NucleraNuclera develops automated benchtop platforms and integrated systems for rapid protein expression, optimization, and purification, utilizing cell-free synthesis, digital microfluidics, and software-driven workflows. Their technology enables miniaturized and scalable protein prototyping—including challenging targets such as membrane proteins—directly at the lab bench. Nuclera serves academic and industrial researchers, focusing on reducing turnaround time for functional protein access and streamlining screening and production. The company has secured significant funding to enable broad commercialization, expanded their leadership team to support scale-up, and continues to drive advancements in drug discovery, proteomics, and experimental automation.
Nuclera develops automated benchtop platforms and integrated systems for rapid protein expression, optimization, and purification, utilizing cell-free synthesis, digital microfluidics, and software-driven workflows. Their technology enables miniaturized and scalable protein prototyping—including challenging targets such as membrane proteins—directly at the lab bench. Nuclera serves academic and industrial researchers, focusing on reducing turnaround time for functional protein access and streamlining screening and production. The company has secured significant funding to enable broad commercialization, expanded their leadership team to support scale-up, and continues to drive advancements in drug discovery, proteomics, and experimental automation.
Publication Number
US-11236377-B2
Publication Date
2022-02-01
Expiration Date
Abstract
The invention relates to the use of specific terminal deoxynucleotidyl transferase (TdT) enzymes in a method of nucleic acid synthesis, to methods of synthesizing nucleic acids, and to the use of kits comprising said enzymes in a method of nucleic acid synthesis. The invention also relates to the use of terminal deoxynucleotidyl transferases and 3′-blocked nucleotide triphosphates in a method of template independent nucleic acid synthesis.
Core Innovation
The invention relates to the use of specific terminal deoxynucleotidyl transferase (TdT) enzymes in a method of nucleic acid synthesis, to methods of synthesizing nucleic acids, and to the use of kits comprising said enzymes in a method of nucleic acid synthesis. The invention also relates to the use of terminal deoxynucleotidyl transferases and 3′-blocked nucleotide triphosphates in a method of template independent nucleic acid synthesis. According to the first aspect, the TdT enzyme comprises an amino acid sequence selected from either any one of SEQ ID NOS: 1 to 5 and 8 or a functional equivalent or fragment thereof having at least 20% sequence homology to said amino acid sequence, or a modified derivative of SEQ ID NO: 6.
The background identifies a problem with current DNA synthesis technology: it is practically impossible to synthesise a DNA strand greater than 200 nucleotides in length using current phosphoramidite chemistry, with most companies offering up to 120 nucleotides, and coupling efficiencies of 95.0-99.5% prevent longer strands in acceptable yields. There is therefore a need to identify terminal deoxynucleotyl transferases that readily incorporate 3′-O reversibly terminated nucleotides and modified said terminal deoxynucleotyl transferases to incorporate 3′-O reversibly terminated nucleotides in a fashion useful for biotechnology and single-stranded DNA synthesis processes.
The inventors located TdT orthologs that have the ability to incorporate deoxynucleotide triphosphates with large 3′-O reversibly terminating moieties and disclose engineered terminal deoxynucleotyl transferases which achieve a substantial increase in incorporation rates of dNTPs containing 3′-O reversibly terminating moieties to be useful for the controlled de novo synthesis of single-stranded DNA. The enzymatic approach is described as having the ability to rapidly produce long lengths of DNA while still maintaining high yields and without using any toxic organic solvents, and the use of inorganic pyrophosphatase is described as reducing build-up of pyrophosphate to reduce backwards reaction and TdT strand dismutation.
Claims Coverage
One independent claim is present. The independent claim recites three main inventive features.
Initiator oligonucleotide immobilized on a solid support
Providing an initiator oligonucleotide immobilized on a solid support.
Addition of a 3′-blocked nucleoside triphosphate
Adding a 3′-blocked nucleoside triphosphate to the initiator in the presence of TdT.
Use of a specific TdT with defined sequence identity
Use of a terminal deoxynucleotidyl transferase (TdT) comprising an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NOS: 1 to 5 and 8, or a functional fragment thereof having terminal deoxynucleotidyl transferase activity, and comprising an N-terminal or a C-terminal truncation.
The independent claim covers a method combining an immobilized initiator oligonucleotide, sequential addition of 3′-blocked nucleoside triphosphates, and the use of a TdT enzyme having at least 95% sequence identity to SEQ ID NOS: 1-5 or 8 (or a functional truncated fragment) to perform template-independent nucleic acid synthesis.
Stated Advantages
Ability to rapidly produce long lengths of DNA while still maintaining high yields.
Avoidance of toxic organic solvents used in current chemical DNA synthesis methods.
Engineered TdT variants achieve substantial increases in incorporation rates of dNTPs containing 3′-O reversibly terminating moieties, enabling controlled de novo single-stranded DNA synthesis.
Use of inorganic pyrophosphatase reduces build-up of pyrophosphate, reducing the rate of the backwards reaction and TdT strand dismutation, thereby improving sequence specificity.
Documented Applications
Gene assembly.
Hybridization microarrays and in situ DNA synthesis for gene assembly or hybridization microarrays.
Introduction of adapter sequences to nucleic acid libraries and library preparation for next-generation sequencing.
Plate or microarray setups and flow instruments (microfluidic or column-based) for nucleic acid synthesis.
Kits comprising TdT and one or more components selected from an initiator sequence, one or more 3′-blocked nucleotide triphosphates, inorganic pyrophosphatase, and a cleaving agent for use in nucleic acid synthesis.
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