Use of terminal transferase enzyme in nucleic acid synthesis
Inventors
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Assignees
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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-11993800-B2
Publication Date
2024-05-28
Expiration Date
Abstract
The invention relates to the use of a modified terminal transferase enzyme in a method of adding one or more nucleotides to the 3′ end of a nucleic acid. The invention also relates to methods of nucleic acid synthesis and sequencing comprising the use of said modified terminal transferase enzyme, to kits comprising said modified terminal transferase enzyme and to the use of said kits in methods of nucleic acid synthesis and sequencing.
Core Innovation
The invention relates to the use of a modified terminal transferase enzyme in a method of adding one or more nucleotides to the 3′ end of a nucleic acid, characterised in that said enzyme comprises a mutated BRCA-1 C-terminal (BRCT) domain. The invention also relates to methods of nucleic acid synthesis and sequencing comprising the use of said modified terminal transferase enzyme, to kits comprising said modified terminal transferase enzyme and to the use of said kits in methods of nucleic acid synthesis and sequencing. The modified terminal transferase enzyme is described as from the DNA polymerase X family, such as terminal deoxynucleotidyl transferase (TdT), and references to TdT include purified and recombinant forms. The invention encompasses the use of said enzyme in methods performed in a microfluidic device and the provision of kits comprising the enzyme together with initiator sequences, reversibly blocked nucleotide triphosphates, inorganic pyrophosphatase, and a cleaving agent.
The background identifies that current DNA synthesis technology cannot practically synthesise DNA strands greater than 200 nucleotides in length using phosphoramidite chemistry and therefore relies on a ‘synthesise and stitch’ technique of overlapping fragments. Known sequencing-by-synthesis methods use template-dependent DNA polymerases and are not suitable for de novo nucleic acid synthesis because of the requirement for an existing template strand. There is therefore a need to provide improved methods of nucleic acid synthesis and sequencing that are able to overcome the problems associated with currently available methods. The invention addresses this need by providing terminal transferase-mediated methods that employ reversibly blocked nucleotide triphosphates and a modified terminal transferase enzyme comprising a mutated or truncated BRCT domain to enable cyclic, sequence-controlled addition of nucleotides.
Claims Coverage
The patent includes two independent claims and four main inventive features extracted from those claims.
Modified terminal transferase with truncated BRCT domain
Use of a modified terminal transferase enzyme characterised in that the enzyme comprises a truncated BRCA-1 C-terminal (BRCT) domain such that the BRCT domain is absent.
Cyclic method of nucleic acid synthesis using reversibly blocked nucleotides
A method comprising providing an initial initiator sequence; adding a reversibly blocked nucleotide triphosphate to the initiator in the presence of the modified terminal transferase; removal of all reagents including the modified terminal transferase; cleaving the blocking group from the added reversibly blocked nucleotide; and removal of the cleaving agent, wherein greater than one nucleotide is added by repeating the adding-to-cleaving cycle.
Modified terminal transferase derived from specified wild-type orthologs
The modified terminal transferase enzyme is derived from a wild type terminal transferase enzyme from L. oculatus, S. harrisii, S. scrofa, O. garnettii, C. lanigera, D. novemcinctus, M. domestica, P. nyererei, or M. brandtii and comprises a truncated BRCT domain.
Kit comprising modified terminal transferase, immobilised initiator and reversibly blocked triphosphates
A kit comprising a modified terminal transferase enzyme (BRCT domain absent) derived from the specified wild type terminal transferases, an immobilized initiator sequence, and one or more reversibly blocked nucleoside triphosphates, optionally in combination with a microfluidic device or chip, inorganic pyrophosphatase, a cleaving agent, and instructions for use in the claimed method.
The independent claims cover (1) a cyclic method of nucleic acid synthesis using a modified terminal transferase lacking a BRCT domain derived from specified orthologs and reversibly blocked nucleotide triphosphates, and (2) a kit that includes the modified terminal transferase, an immobilized initiator sequence and reversibly blocked nucleoside triphosphates, with optional components for use in the method.
Stated Advantages
N-terminal truncations of TdT endow wild-type and engineered TdTs with much higher cycling efficiencies that greatly enhance the industrial utility of TdT-mediated nucleic acid synthesis.
BRCT truncation increases solubility and surface accessibility and prevents surface fouling, protein aggregation and general protein misbehavior, enabling efficient multi-cycle addition of nucleotides to surface immobilized DNA.
Modified TdT variants lacking the BRCT domain are capable of adding modified nucleotide triphosphates quantitatively in series, enabling multi-step de novo enzymatic DNA synthesis and the addition of multiple reversibly blocked nucleotides in series.
The use of an inorganic pyrophosphatase reduces the build-up of pyrophosphate and has the advantage of reducing the rate of the backwards reaction and terminal transferase strand dismutation.
The method provides a novel way to introduce adapter sequences to a nucleic acid library, offering an improved approach to library preparation compared to single-stranded ligation.
Documented Applications
Methods of nucleic acid synthesis and sequencing comprising the use of a modified terminal transferase enzyme with a mutated or truncated BRCT domain.
De novo enzymatic DNA or RNA strand synthesis by cyclic addition and deprotection of reversibly blocked nucleotide triphosphates to an initiator sequence, including performance in a microfluidic device or flow instrument.
Kits comprising the modified terminal transferase enzyme, an initiator sequence, reversibly blocked nucleotide triphosphates, optionally a microfluidic device, inorganic pyrophosphatase and a cleaving agent for use in nucleic acid synthesis or sequencing.
Alternating-phase nucleic acid synthesis processes and alternating-phase polymer synthesis methods that couple immobilised monomers/dNTPs to polymers/initiator sequences via the modified terminal transferase and cleavable linkers.
Installation of adapter sequences to nucleic acid libraries (library preparation for next-generation sequencing) by adding defined nucleic acid sequences to initiator fragments.
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