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-11807887-B2
Publication Date
2023-11-07
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 invention, TdT enzymes comprising an amino acid sequence selected from either: (a) 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 (b) a modified derivative of SEQ ID NO: 6 are used in a method of nucleic acid synthesis. The method is described conceptually as providing an initiator sequence, adding a 3′-blocked nucleotide triphosphate to the initiator sequence in the presence of a TdT as defined, removing reagents from the initiator sequence, cleaving the blocking group from the 3′-blocked nucleotide triphosphate in the presence of a cleaving agent, and removing the cleaving agent.
The background problem the invention addresses is that current DNA synthesis technology is practically impossible to synthesise a DNA strand greater than 120-200 nucleotides in length due to the stepwise efficiency limits of phosphoramidite chemistry, and that commercially available recombinant Bos taurus TdT is unable to add 3′-O reversibly terminated nucleotide triphosphates in a quantitative fashion. The invention identifies TdT orthologs and engineered terminal deoxynucleotidyl transferases that readily incorporate 3′-O reversibly terminated nucleotides and engineered variants which achieve a substantial increase in incorporation rates of such dNTPs to enable controlled de novo synthesis of single-stranded DNA.
Claims Coverage
The independent claim (claim 1) recites two main inventive features.
TdT comprising specified amino acid sequences
A terminal deoxynucleotidyl transferase (TdT) comprising an amino acid sequence of 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.
Combination with one or more 3′-blocked nucleoside triphosphates
The kit is in combination with one or more 3′-blocked nucleoside triphosphates.
Claim 1 covers a kit combining a TdT having at least 95% sequence identity to any one of SEQ ID NOS: 1-5 and 8 (or a functional N- or C-terminally truncated fragment) together with one or more 3′-blocked nucleoside triphosphates.
Stated Advantages
Ability to rapidly produce long lengths of DNA while still maintaining high yields.
Avoids using toxic organic solvents.
Enzymatic method can produce DNA strands beyond the 120-200 nucleotide limit of current synthetic DNA synthesis methods.
Use of inorganic pyrophosphatase reduces build-up of pyrophosphate, thereby reducing the rate of backwards reaction and TdT strand dismutation.
Documented Applications
Gene assembly.
Hybridization microarrays.
Introduction of adapter sequences to a nucleic acid library and library preparation for next-generation sequencing.
Use in a kit for nucleic acid synthesis comprising TdT, one or more 3′-blocked nucleotide triphosphates, inorganic pyrophosphatase and a cleaving agent.
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