Modified terminal deoxynucleotidyl transferase (TdT) enzymes
Inventors
Chen, Michael Chun Hao • Mcinroy, Gordon Ross • Chen, Sihong • Cook, Ian Haston
Assignees
Publication Number
US-12312611-B2
Publication Date
2025-05-27
Expiration Date
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
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 their use 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 new terminal deoxynucleotidyl transferases and 3′-blocked nucleoside triphosphates in a method of template independent nucleic acid synthesis.
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 their use 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 new terminal deoxynucleotidyl transferases and 3′-blocked nucleoside triphosphates in a method of template independent nucleic acid synthesis.
Current DNA synthesis technology does not meet the demands of the biotechnology industry because it is practically impossible to synthesise a DNA strand greater than 200 nucleotides in length using current phosphoramidite chemistry, and known methods cannot efficiently incorporate 3′-O—reversibly terminating moieties using terminal deoxynucleotidyl transferase (TdT). There is therefore a need to identify modified terminal deoxynucleotidyl transferases that readily incorporate 3′-O—reversibly terminated nucleotides in a fashion useful for biotechnology and single-stranded DNA synthesis processes.
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 homologous amino acid sequences of TdT or family X or A polymerases of other species. The patent discloses that certain modifications improve solubility and handling of the enzyme and that other modifications improve the ability to incorporate nucleotides with modifications, including modifications at the 3′-position of the sugar and modifications to the base, and that the corresponding modifications can be introduced into homologous sequences from other species.
Claims Coverage
This patent specification contains two independent claims directed to (1) a modified terminal deoxynucleotidyl transferase and (2) a method of nucleic acid synthesis using a modified TdT. The main inventive features are summarized below.
Modified terminal deoxynucleotidyl transferase with specified amino acid modifications
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 SEQ ID NO 1, and wherein said modified TdT or said truncated version thereof differs from said wild type TdT with amino acid modifications at one or more of the amino acids: V32, A33, I34, F35, A53, V68, V71, E97, I101, M108, G109, A110, Q115, V116, S125, T137, Q143, M152, E153, N154, H155, N156, Q157, I158, I165, N169, N173, S175, E176, G177, P178, C179, L180, A181, F182, M183, R184, A185, L188, H194, A195, I196, S197, S198, S199, K200, E203, G204, D210, Q211, T212, K213, A214, I216, E217, D218, L220, Y222, V228, D230, Q238, T239, L242, L251, K260, G261, F262, H263, S264, L265, E267, Q269, A270, D271, N272, A273, H275, F276, T277, K278, M279, Q280, K281, S291, A292, A293, V294, C295, K296, E298, A299, Q300, A301, Q304, I305, T309, V310, R311, L312, I313, A314, I318, V319, T320, G328, K329, E330, C331, L338, T341, P342, E343, M344, G345, K346, W349, L350, L351, N352, R353, L354, I355, N356, R357, L358, Q359, N360, Q361, G362, I363, L364, L365, Y366, Y367, D368, I369, V370, K376, T377, C381, K383, D388, H389, F390, Q391, K392, F394, I397, K398, K400, K401, E402, L403, A404, A405, G406, R407, D411, A421, P422, P423, V424, D425, N426, F427, A430, R438, F447, A448, R449, H450, E451, R452, K453, M454, L455, L456, D457, N458, H459, A460, L461, Y462, D463, K464, T465, K466, K467, T474, D477, D485, Y486, I487, D488, P489.
Method of nucleic acid synthesis using a modified TdT and 3′-blocked nucleotides
A method of nucleic acid synthesis comprising use of an initiator oligonucleotide, a nucleoside triphosphate having a 3′-blocking group, and the modified terminal deoxynucleotidyl transferase or truncated version thereof as defined in the patent; the method includes cyclical addition of a 3′-blocked nucleoside triphosphate to the initiator in the presence of the modified TdT, removal of reagents, cleaving the 3′-blocking group in the presence of a cleaving agent, and removal of the cleaving agent [procedural detail omitted for safety].
Claims cover (1) modified TdT enzymes differing from SEQ ID NO 1 by amino acid modifications at an extensive list of specified residues or regions, and (2) a method of nucleic acid synthesis that uses 3′-blocked nucleoside triphosphates together with the modified TdT in a cyclical addition-and-deprotection workflow.
Stated Advantages
Improved ability to incorporate nucleotides with modifications, including modifications at the 3′-position of the sugar and modifications to the base.
Improved solubility and handling of the enzyme attributable to modifications within defined amino acid regions.
Increased TdT incorporation activity of reversibly terminated nucleotides and removal of substrate biases during incorporation of modified nucleoside triphosphates.
Enables controlled single-stranded DNA synthesis useful for biotechnology applications such as in situ DNA synthesis for gene assembly or hybridization microarrays by removing the need for an anhydrous environment and allowing the use of various polymers incompatible with organic solvents.
Documented Applications
Use of engineered TdT enzymes or homologous X family/A family polymerases in methods of nucleic acid synthesis and in methods of synthesizing nucleic acids.
Use of kits comprising said enzymes in a method of nucleic acid synthesis.
Template independent nucleic acid synthesis using new terminal deoxynucleotidyl transferases and 3′-blocked nucleoside triphosphates.
De novo single-stranded DNA synthesis and in situ DNA synthesis for gene assembly or hybridization microarrays.
Preparation of homopolymeric adaptor sequences and sequencing library preparation for next-generation sequencing.
Solid-support synthesis of defined nucleic acid sequences using cycles of blocked-nucleotide addition and deprotection.
Extension of disclosed motifs, regions, and mutations to homologous family X polymerases and A family polymerases (including Polθ) to enable modified polymerases to incorporate 3′-modified nucleotides for nucleic acid synthesis applications.
Provision of codon-optimized nucleic acid sequences and cell lines producing the modified terminal transferase for expression and use.
Interested in licensing this patent?