Engineered Terminal Deoxynucleotidyl Transferase Variants
AssigneeCodexis, Inc.
InventorsDavid Entwistle, Stephanie Forget, Michelle Li, Niusha Mahmoodi, Ryan Reeves, Ljubica Vojcic, Jonathan Vroom, David Watts, Leland Ken Wong
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Abstract
The present invention provides engineered terminal deoxynucleotidyl transferase (TdT) polypeptides useful in template-independent polynucleotide synthesis, as well as compositions, methods of utilizing these engineered polypeptides, and polynucleotides encoding the engineered terminal deoxynucleotidyl transferases.
Claims97
An engineered terminal deoxynucleotidyl transferase comprising a polypeptide sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence selected from SEQ ID NO:
- 2, 8, 12, 16, 24, 36, 54, 268, 648, 660, 882, 1100, 1336, 1348, 1596, 1654, 1830, 1950, 2008, 2254, 2514, 2524, 2638, 2804, 2812, 2956, 3174, 3222, 3670, 3674, 3796, 3870, 3918, 4266, 4442, 4654, 4850, 4856, 4904, 5002, 5028, 5192, 5246, or a functional fragment thereof, and at least one amino acid residue difference relative to the reference sequence.
A polynucleotide sequence comprising at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence of SEQ ID NOs:
- 1, 7, 11, 15, 23, 35, 53, 267, 647, 659, 881, 1099, 1335, 1347, 1595, 1653, 1829, 1949, 2007, 2253, 2513, 2523, 2637, 2803, 2811, 2955, 3173, 3221, 3669, 3673, 3795, 3869, 3917, 4265, 4441, 4653, 4849, 4855, 4903, 5001, 5027, 5191, and 5245, and/or or a functional fragment thereof, wherein said polynucleotide sequence encodes an engineered polypeptide comprising at least one substitution at one or more amino acid positions.
A method for template-independent synthesis of an oligonucleotide, the method comprising:
- a) providing at least one terminal deoxynucleotidyl transferase or template-independent polymerase;
- b) providing at least one oligo acceptor substrate, wherein the oligo acceptor substrate comprises a 3′-OH, or an equivalent thereof;
- c) contacting the oligo acceptor substrate, the terminal deoxynucleotidyl transferase or template-independent polymerase, and one or more nucleotide triphosphates, modified nucleotide triphosphates, or NTP-3′-O-RBG under conditions sufficient for the addition of the nucleotide, modified nucleotide, or nucleotide-3′-O-RBG to the 3′ end of the oligo acceptor substrate.
Dependencies are inferred from the claim text.