Modified terminal deoxynucleotidyl transferase (TdT) enzymes
Inventors
Chen, Michael Chun Hao • Chen, Sihong • Mcinroy, Gordon Ross
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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-12116600-B2
Publication Date
2024-10-15
Expiration Date
Abstract
The invention relates to engineered terminal deoxynucleotidyl transferase (TdT) enzymes or the homologous amino acid sequence of Polμ, Polβ, Polλ, and Polθ of any species or the homologous amino acid sequence of X family polymerases of any species and uses thereof.
Core Innovation
The invention relates to engineered terminal deoxynucleotidyl transferase (TdT) enzymes or the homologous amino acid sequence of Polμ, Polβ, Polλ, and Polθ of any species or the homologous amino acid sequence of X family polymerases of any species and uses thereof. Described herein are modified terminal deoxynucleotidyl transferase (TdT) enzymes comprising amino acid modifications when compared to a wild type sequence SEQ ID NO: 1 or a truncated version thereof or the homologous amino acid sequence of a terminal deoxynucleotidyl transferase (TdT) enzyme in other species or the homologous amino acid sequence of Polμ, Polβ, Polλ, and Polθ of any species or the homologous amino acid sequence of X family polymerases of any species. Amino acid locations having improved activity include R193, E456, R457, or N473. Modifications include one or more of R193H, E456G, R457S, and/or N473G, including combinations such as E456G and N473G, and sequences comprising at least SEQ ID NO: 3 or truncated versions thereof wherein the sequence has one or more amino acid modifications in one or more of the amino acid positions R193, E456, R457, and/or N473 of the full length sequence.
The background describes that current DNA synthesis technology does not meet the demands of the biotechnology industry because it is highly challenging to synthesise DNA strands greater than 120-200 nucleotides in viable yield using phosphoramidite chemistry, and that there is a need to identify modified terminal deoxynucleotidyl transferases that readily incorporate 3′-O-reversibly terminated nucleotides. The modifications described herein improve the ability to incorporate native nucleotides, particularly dATP, the enzymes have a higher catalytic activity than the unmodified enzymes and operate at higher temperatures, and the enzymes are especially well suited to terminator-free enzymatic DNA synthesis for de novo template-independent nucleic acid synthesis.
Claims Coverage
Independent claim 1 defines a modified terminal deoxynucleotyl transferase (TdT) or a truncated version retaining TdT activity and identifies three main inventive features.
Modified TdT or truncated version retaining activity
A modified terminal deoxynucleotidyl transferase (TdT) of a wild type TdT, or a truncated version of said modified TdT that retains TdT activity, wherein the wild type TdT has the amino acid sequence of SEQ ID NO: 1.
Modification at residue corresponding to R193
A modification at a residue corresponding to R193 of SEQ ID NO: 1.
Modifications at residues corresponding to E456, R457 and N473
A modification at two or more amino acid residue positions corresponding to E456, R457 and N473 of SEQ ID NO: 1.
Claim 1 covers a modified TdT (including truncated versions that retain activity) distinguished by amino acid modifications at residue positions corresponding to R193 and/or two or more of E456, R457, and N473 of SEQ ID NO: 1.
Stated Advantages
Improved ability to incorporate native nucleotides, particularly dATP.
Higher catalytic activity and greater catalytic efficiency than unmodified enzymes.
Operation at higher temperatures and enhanced thermostability compared to parent enzymes.
Utilisation of nucleotide reagents at lower concentration than wild type sequences.
Suitability for terminator-free enzymatic DNA synthesis and de novo template-independent nucleic acid synthesis.
Capability to preferentially incorporate 3′-OH nucleotides to deplete native reagents and improve synthesis efficiency.
Use in other molecular biology applications including the TUNEL assay.
Documented Applications
Methods of nucleic acid synthesis, including adding nucleotides to an initiator oligonucleotide in the presence of the modified TdT (method claim).
De novo template-independent nucleic acid synthesis and terminator-free enzymatic DNA synthesis.
Kits comprising a terminal deoxynucleotidyl transferase (TdT) as defined herein in combination with an initiator sequence and one or more nucleoside triphosphates (for example dATP).
Use to remove nucleotides containing a 3′-OH moiety in a solution of nucleotides containing a 3′-block or 3′-reversible block or 3′-terminator moiety.
In situ DNA synthesis for gene assembly or hybridization microarrays (as described in the background).
Use in the TUNEL assay for identifying and quantifying apoptotic cells.
Provision of the nucleic acid sequence and codon-optimized cDNA sequences to express the modified TdT and a cell line producing the modified terminal transferase.
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