Thermostable terminal deoxynucleotidyl transferase

Abstract

Disclosed herein is a recombinant terminal deoxynucleotidyl transferase (TdT). In some embodiments, the recombinant TdT comprises an amino acid sequence at least 80% identical to bovine TdT, wherein the recombinant TdT comprises one or more amino acid substitution mutations at one or more positions functionally equivalent to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 of bovine TdT.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 023,734, filed May 12, 2020, the entire contents of which are incorporated herein by reference in their entirety.

[0002] (Reference to sequence listing) This application has been filed with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled Sequences_Listing_47CX-311971-WO (created May 11, 2021, 56 Kb in size). The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

[0003] FIELD OF THE INVENTION The present disclosure relates generally to the field of recombinant proteins, for example, recombinant terminal deoxynucleotidyl transferase. [Background technology]

[0004] Terminal deoxynucleotidyl transferase (TdT) catalyzes the template-free incorporation of any nucleotide into single-stranded DNA. However, wild-type (WT) TdT is not optimized for the incorporation of 3'-modified nucleotides. Furthermore, TdT is only marginally stable, and evolution for the ability to incorporate 3'-block nucleotides is likely to lead to unstable mutants, as most mutations result in reduced thermostability. Therefore, there is a need for thermostable TdT variants that can serve as a starting point for the generation of mutants that enable the efficient incorporation of 3'-modified nucleotides. Summary of the Invention

[0005] Disclosed herein are embodiments of recombinant terminal deoxynucleotidyl transferase (TdT). In some embodiments, the recombinant TdT comprises an amino acid sequence at least 80% identical to SEQ ID NO: 1, and the recombinant TdT comprises one or more amino acid substitution mutations at one or more positions functionally equivalent to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 of Bos taurus TdT of SEQ ID NO: 12.

[0006] In some embodiments, the amino acid substitution mutation at a position functionally equivalent to Glu191 of Bos taurus TdT of SEQ ID NO: 12 comprises a mutation to a nonpolar amino acid, a polar amino acid, a positively charged amino acid, a negatively charged amino acid, a hydrophobic amino acid, an aromatic amino acid, an aliphatic amino acid, a small amino acid, or a hydrophilic amino acid. The amino acid substitution mutation at a position functionally equivalent to Glu191 can comprise a mutation to a nonpolar amino acid, a hydrophobic amino acid, an aliphatic amino acid, or a branched-chain amino acid. An amino acid substitution mutation at a position functionally equivalent to Glu191 of Bos taurus TdT of SEQ ID NO: 12 can be Glu191Ala, Glu191Arg, Glu191Asn, Glu191Asp, Glu191Cys, Glu191Gln, Glu191Gly, Glu191His, Glu191Ile, Glu191Leu, Glu191Lys, Glu191Met, Glu191Phe, Glu191Pro, Glu191Ser, Glu191Thr, Glu191Trp, Glu191Tyr, or Glu191Val. An amino acid substitution mutation at a position functionally equivalent to Glu191 of Bos taurus TdT of SEQ ID NO: 12 can be Glu191Ala, Glu191Gly, Glu191Ile, Glu191Leu, Glu191Met, or Glu191Val. An amino acid substitution mutation at a position functionally equivalent to Glu191 of Bos taurus TdT of SEQ ID NO: 12 can be Glu191Val.

[0007] In some embodiments, the amino acid substitution mutation at a position functionally equivalent to Lys193 of Bos taurus TdT of SEQ ID NO: 12 comprises a mutation to a nonpolar amino acid, a polar amino acid, a positively charged amino acid, a negatively charged amino acid, a hydrophobic amino acid, an aromatic amino acid, an aliphatic amino acid, a small amino acid, or a hydrophilic amino acid. The amino acid substitution mutation at a position functionally equivalent to Lys193 of Bos taurus TdT of SEQ ID NO: 12 can comprise a mutation to a polar amino acid or a hydrophilic amino acid. The amino acid substitution mutation at a position functionally equivalent to Lys193 of Bos taurus TdT of SEQ ID NO: 12 can be Lys193Ala, Lys193Arg, Lys193Asn, Lys193Asp, Lys193Cys, Lys193Gln, Lys193Glu, Lys193Gly, Lys193His, Lys193Ile, Lys193Leu, Lys193Met, Lys193Phe, Lys193Pro, Lys193Ser, Lys193Thr, Lys193Trp, Lys193Tyr, or Lys193Val. The amino acid substitution mutation at a position functionally equivalent to Lys193 of Bos taurus TdT of SEQ ID NO: 12 can be Lys193Asn, Lys193Gln, Lys193Ser, or Lys193Thr. The amino acid substitution mutation at a position functionally equivalent to Lys193 of Bos taurus TdT of SEQ ID NO: 12 can be Lys193Asn.

[0008] In some embodiments, the amino acid substitution mutation at a position functionally equivalent to Glu194 of Bos taurus TdT of SEQ ID NO: 12 comprises a mutation to a nonpolar amino acid, a polar amino acid, a positively charged amino acid, a negatively charged amino acid, a hydrophobic amino acid, an aromatic amino acid, an aliphatic amino acid, a small amino acid, or a hydrophilic amino acid. The amino acid substitution mutation at a position functionally equivalent to Glu194 of Bos taurus TdT of SEQ ID NO: 12 can comprise a mutation to a nonpolar amino acid, a hydrophobic amino acid, an aliphatic amino acid, or a branched-chain amino acid. An amino acid substitution mutation at a position functionally equivalent to Glu194 of Bos taurus TdT of SEQ ID NO: 12 can be Glu194Ala, Glu194Arg, Glu194Asn, Glu194Asp, Glu194Cys, Glu194Gln, Glu194Gly, Glu194His, Glu194Ile, Glu194Leu, Glu194Lys, Glu194Met, Glu194Phe, Glu194Pro, Glu194Ser, Glu194Thr, Glu194Trp, Glu194Tyr, or Glu194Val. An amino acid substitution mutation at a position functionally equivalent to Glu194 of Bos taurus TdT of SEQ ID NO: 12 can be Glu194Ala, Glu194Gly, Glu194Ile, Glu194Leu, Glu194Met, or Glu194Val. An amino acid substitution mutation at a position functionally equivalent to Glu194 of Bos taurus TdT of SEQ ID NO: 12 can be Glu194Gly.

[0009] In some embodiments, the amino acid substitution mutation at a position functionally equivalent to Asp242 of Bos taurus TdT of SEQ ID NO: 12 comprises a mutation to a nonpolar amino acid, a polar amino acid, a positively charged amino acid, a negatively charged amino acid, a hydrophobic amino acid, an aromatic amino acid, an aliphatic amino acid, a small amino acid, or a hydrophilic amino acid. The amino acid substitution mutation at a position functionally equivalent to Asp242 of Bos taurus TdT of SEQ ID NO: 12 can comprise a mutation to a polar amino acid or an aromatic amino acid. The amino acid substitution mutation at a position functionally equivalent to Asp242 of Bos taurus TdT of SEQ ID NO: 12 can be Asp242Ala, Asp242Arg, Asp242Asn, Asp242Cys, Asp242Gln, Asp242Glu, Asp242Gly, Asp242His, Asp242Ile, Asp242Leu, Asp242Lys, Asp242Met, Asp242Phe, Asp242Pro, Asp242Ser, Asp242Thr, Asp242Trp, Asp242Tyr, or Asp242Val. The amino acid substitution mutation at a position functionally equivalent to Asp242 of Bos taurus TdT of SEQ ID NO: 12 can be Asp242Asn, Asp242Gln, Asp242Phe, Asp242Ser, Asp242Thr, Asp242Trp, or Asp242Tyr. The amino acid substitution mutation at a position functionally equivalent to Asp242 of Bos taurus TdT of SEQ ID NO: 12 can be Asp242Tyr.

[0010] In some embodiments, the amino acid substitution mutation at a position functionally equivalent to Lys287 of Bos taurus TdT of SEQ ID NO: 12 comprises a mutation to a nonpolar amino acid, a polar amino acid, a positively charged amino acid, a negatively charged amino acid, a hydrophobic amino acid, an aromatic amino acid, an aliphatic amino acid, a small amino acid, or a hydrophilic amino acid. The amino acid substitution mutation at a position functionally equivalent to Lys287 of Bos taurus TdT of SEQ ID NO: 12 can comprise a mutation to a polar amino acid, a negatively charged amino acid, or a hydrophilic amino acid. The amino acid substitution mutation at a position functionally equivalent to Lys287 of Bos taurus TdT of SEQ ID NO: 12 can be Lys287Ala, Lys287Arg, Lys287Asn, Lys287Asp, Lys287Cys, Lys287Gln, Lys287Glu, Lys287Gly, Lys287His, Lys287Ile, Lys287Leu, Lys287Met, Lys287Phe, Lys287Pro, Lys287Ser, Lys287Thr, Lys287Trp, Lys287Tyr, or Lys287Val. The amino acid substitution mutation at a position functionally equivalent to Lys287 of Bos taurus TdT of SEQ ID NO: 12 can be Lys287Asp or Lys287Glu. The amino acid substitution mutation at a position functionally equivalent to Lys287 of Bos taurus TdT of SEQ ID NO: 12 can be Lys287Glu.

[0011] In some embodiments, the amino acid substitution mutation at a position functionally equivalent to Phe296 of Bos taurus TdT of SEQ ID NO: 12 comprises a mutation to a nonpolar amino acid, a polar amino acid, a positively charged amino acid, a negatively charged amino acid, a hydrophobic amino acid, an aromatic amino acid, an aliphatic amino acid, a small amino acid, or a hydrophilic amino acid. The amino acid substitution mutation at a position functionally equivalent to Phe296 of Bos taurus TdT of SEQ ID NO: 12 can comprise a mutation to a nonpolar amino acid, a hydrophobic amino acid, an aliphatic amino acid, or a branched-chain amino acid. The amino acid substitution mutation at a position functionally equivalent to Phe296 of Bos taurus TdT of SEQ ID NO: 12 can be Phe296Ala, Phe296Arg, Phe296Asn, Phe296Asp, Phe296Cys, Phe296Gln, Phe296Glu, Phe296Gly, Phe296His, Phe296Ile, Phe296Leu, Phe296Lys, Phe296Met, Phe296Pro, Phe296Ser, Phe296Thr, Phe296Trp, Phe296Tyr, or Phe296Val. The amino acid substitution mutation at a position functionally equivalent to Phe296 of Bos taurus TdT of SEQ ID NO: 12 can be Phe296Ala, Phe296Gly, Phe296Ile, Phe296Leu, Phe296Met, or Phe296Val. The amino acid substitution mutation at a position functionally equivalent to Phe296 of Bos taurus TdT of SEQ ID NO: 12 can be Phe296Leu.

[0012] In some embodiments, the amino acid substitution mutation at a position functionally equivalent to Met299 of Bos taurus TdT of SEQ ID NO: 12 comprises a mutation to a nonpolar amino acid, a polar amino acid, a positively charged amino acid, a negatively charged amino acid, a hydrophobic amino acid, an aromatic amino acid, an aliphatic amino acid, a small amino acid, or a hydrophilic amino acid. The amino acid substitution mutation at a position functionally equivalent to Met299 of Bos taurus TdT of SEQ ID NO: 12 can comprise a mutation to a polar amino acid, a positively charged amino acid, or a hydrophilic amino acid. The amino acid substitution mutation at a position functionally equivalent to Met299 of Bos taurus TdT of SEQ ID NO: 12 can be Met299Ala, Met299Arg, Met299Asn, Met299Asp, Met299Cys, Met299Gln, Met299Glu, Met299Gly, Met299His, Met299Ile, Met299Leu, Met299Lys, Met299Phe, Met299Pro, Met299Ser, Met299Thr, Met299Trp, Met299Tyr, or Met299Val. The amino acid substitution mutation at a position functionally equivalent to Met299 of Bos taurus TdT of SEQ ID NO: 12 can be Met299Arg, Met299His, or Met299Lys. The amino acid substitution mutation at a position functionally equivalent to Met299 of Bos taurus TdT of SEQ ID NO: 12 can be Met299Lys.

[0013] In some embodiments, the amino acid substitution mutation at a position functionally equivalent to Thr342 of Bos taurus TdT of SEQ ID NO: 12 comprises a mutation to a nonpolar amino acid, a polar amino acid, a positively charged amino acid, a negatively charged amino acid, a hydrophobic amino acid, an aromatic amino acid, an aliphatic amino acid, a small amino acid, or a hydrophilic amino acid. The amino acid substitution mutation at a position functionally equivalent to Thr342 of Bos taurus TdT of SEQ ID NO: 12 can comprise a mutation to a polar amino acid, an aliphatic amino acid, or a hydrophilic amino acid. The amino acid substitution mutation at a position functionally equivalent to Thr342 of Bos taurus TdT of SEQ ID NO: 12 can be Thr342Ala, Thr342Arg, Thr342Asn, Thr342Asp, Thr342Cys, Thr342Gln, Thr342Glu, Thr342Gly, Thr342His, Thr342Ile, Thr342Leu, Thr342Lys, Thr342Met, Thr342Phe, Thr342Pro, Thr342Ser, Thr342Trp, Thr342Tyr, or Thr342Val. The amino acid substitution mutation at a position functionally equivalent to Thr342 of Bos taurus TdT of SEQ ID NO: 12 can be Thr342Asn, Thr342Cys, Thr342Gln, Thr342Pro, or Thr342Ser. An amino acid substitution mutation at a position functionally equivalent to Thr342 of Bos taurus TdT of SEQ ID NO: 12 can be Thr342Ser.

[0014] In some embodiments, the amino acid substitution mutation at a position functionally equivalent to His421 of Bos taurus TdT of SEQ ID NO: 12 comprises a mutation to a nonpolar amino acid, a polar amino acid, a positively charged amino acid, a negatively charged amino acid, a hydrophobic amino acid, an aromatic amino acid, an aliphatic amino acid, a small amino acid, or a hydrophilic amino acid. The amino acid substitution mutation at a position functionally equivalent to His421 of Bos taurus TdT of SEQ ID NO: 12 can comprise a mutation to a polar amino acid, an aliphatic amino acid, or a hydrophilic amino acid. The amino acid substitution mutation at a position functionally equivalent to His421 of Bos taurus TdT of SEQ ID NO: 12 can be His421Ala, His421Arg, His421Asn, His421Asp, His421Cys, His421Gln, His421Glu, His421Gly, His421Ile, His421Leu, His421Lys, His421Met, His421Phe, His421Pro, His421Ser, His421Thr, His421Trp, His421Tyr, or His421Val. The amino acid substitution mutation at a position functionally equivalent to His421 of Bos taurus TdT of SEQ ID NO: 12 can be His421Asn, His421Cys, His421Gln, His421Pro, His421Ser, or His421Thr. The amino acid substitution mutation at a position functionally equivalent to His421 of Bos taurus TdT of SEQ ID NO: 12 can be His421Pro.

[0015] In some embodiments, the recombinant TdT comprises two or more amino acid substitution mutations at two or more positions functionally equivalent to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 of Bos taurus TdT of SEQ ID NO: 12. Two or more amino acid substitution mutations at two or more positions functionally equivalent to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 of Bos taurus TdT of SEQ ID NO: 12 can include two or more of Glu191Val, Lys193Asn, Glu194Gly, Asp242Tyr, Lys287Glu, Phe296Leu, Met299Lys, Thr342Ser, and His421Pro, respectively.

[0016] In some embodiments, the recombinant TdT comprises three or more amino acid substitution mutations at three or more positions functionally equivalent to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 of Bos taurus TdT of SEQ ID NO: 12. The three or more amino acid substitution mutations at three or more positions functionally equivalent to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 of Bos taurus TdT of SEQ ID NO: 12 can include three or more of Glu191Val, Lys193Asn, Glu194Gly, Asp242Tyr, Lys287Glu, Phe296Leu, Met299Lys, Thr342Ser, and His421Pro, respectively.

[0017] In some embodiments, the recombinant TdT comprises four or more amino acid substitution mutations at four or more positions functionally equivalent to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 of Bos taurus TdT of SEQ ID NO: 12. The four or more amino acid substitution mutations at four or more positions functionally equivalent to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 of Bos taurus TdT of SEQ ID NO: 12 can include four or more of Glu191Val, Lys193Asn, Glu194Gly, Asp242Tyr, Lys287Glu, Phe296Leu, Met299Lys, Thr342Ser, and His421Pro, respectively.

[0018] In some embodiments, the recombinant TdT comprises five or more amino acid substitution mutations at five or more positions functionally equivalent to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 of Bos taurus TdT of SEQ ID NO: 12. The five or more amino acid substitution mutations at five or more positions functionally equivalent to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 of Bos taurus TdT of SEQ ID NO: 12 can include five or more of Glu191Val, Lys193Asn, Glu194Gly, Asp242Tyr, Lys287Glu, Phe296Leu, Met299Lys, Thr342Ser, and His421Pro, respectively.

[0019] In some embodiments, the recombinant TdT comprises six or more amino acid substitution mutations at six or more positions functionally equivalent to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 of Bos taurus TdT of SEQ ID NO: 12. The six or more amino acid substitution mutations at six or more positions functionally equivalent to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 of Bos taurus TdT of SEQ ID NO: 12 can include six or more of Glu191Val, Lys193Asn, Glu194Gly, Asp242Tyr, Lys287Glu, Phe296Leu, Met299Lys, Thr342Ser, and His421Pro, respectively.

[0020] In some embodiments, the recombinant TdT comprises seven or more amino acid substitution mutations at seven or more positions functionally equivalent to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 of Bos taurus TdT of SEQ ID NO: 12. The seven or more amino acid substitution mutations at seven or more positions functionally equivalent to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 of Bos taurus TdT of SEQ ID NO: 12 can include seven or more of Glu191Val, Lys193Asn, Glu194Gly, Asp242Tyr, Lys287Glu, Phe296Leu, Met299Lys, Thr342Ser, and His421Pro, respectively.

[0021] In some embodiments, the recombinant TdT comprises eight or more amino acid substitution mutations at eight or more positions functionally equivalent to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 of Bos taurus TdT of SEQ ID NO: 12. The eight or more amino acid substitution mutations at eight or more positions functionally equivalent to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 of Bos taurus TdT of SEQ ID NO: 12 can include eight or more of Glu191Val, Lys193Asn, Glu194Gly, Asp242Tyr, Lys287Glu, Phe296Leu, Met299Lys, Thr342Ser, and His421Pro, respectively.

[0022] In some embodiments, the recombinant TdT comprises eight amino acid substitution mutations at eight positions functionally equivalent to Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 of Bos taurus TdT of SEQ ID NO: 12. The eight amino acid substitution mutations at eight positions functionally equivalent to Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 of Bos taurus TdT of SEQ ID NO: 12 can be Lys193Asn, Glu194Gly, Asp242Tyr, Lys287Glu, Phe296Leu, Met299Lys, Thr342Ser, and His421Pro, respectively.

[0023] In some embodiments, the recombinant TdT comprises nine amino acid substitution mutations at positions functionally equivalent to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 of Bos taurus TdT of SEQ ID NO: 12. The nine amino acid substitution mutations at positions functionally equivalent to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 of Bos taurus TdT of SEQ ID NO: 12 can be Glu191Val, Lys193Asn, Glu194Gly, Asp242Tyr, Lys287Glu, Phe296Leu, Met299Lys, Thr342Ser, and His421Pro, respectively.

[0024] In some embodiments, the recombinant TdT comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 1. The recombinant TdT comprises an amino acid sequence that may be at least 90% identical to SEQ ID NO: 1. The recombinant TdT comprises an amino acid sequence that may be at least 95% identical to SEQ ID NO: 1. The recombinant TdT comprises an amino acid sequence that may be at least 95% identical to SEQ ID NO: 11. The recombinant TdT comprises an amino acid sequence that may be at least 80% identical to SEQ ID NO: 12.

[0025] In some embodiments, the recombinant TdT is stable at temperatures of 47°C or higher. The recombinant TdT may be stable at temperatures of 50°C or higher. The recombinant TdT may be stable at temperatures of 55°C or higher. The recombinant TdT may be stable at temperatures of 58°C or higher. In some embodiments, the terminal deoxynucleotidyl transferase activity of the recombinant TdT is at least 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, or 120% of the terminal deoxynucleotidyl transferase activity of Bos taurus TdT of SEQ ID NO: 12 at the same test temperature. The test temperature can be 37°C, 47°C, 50°C, 55°C, or 58°C.

[0026] In some embodiments, the recombinant TdT comprises a small ubiquitin-like modifier (SUMO) fragment. The SUMO fragment comprises an amino acid sequence that may be at least 80% identical to SEQ ID NO: 13. The recombinant TdT may comprise a SUMO fragment at the N-terminus of the recombinant TdT. The recombinant TdT may comprise an amino acid sequence that is at least 80% identical to SEQ ID NO: 14. The recombinant TdT may comprise an amino acid sequence that is at least 80% identical to SEQ ID NO: 15. The recombinant TdT may comprise a SUMO fragment at the C-terminus of the recombinant TdT.

[0027] Disclosed herein are embodiments of polynucleotides encoding the recombinant terminal deoxynucleotidyl transferase (TdT) of the present disclosure. Disclosed herein are embodiments of expression vectors comprising a polynucleotide encoding the recombinant terminal deoxynucleotidyl transferase (TdT) of the present disclosure. Disclosed herein are embodiments of host cells comprising the recombinant terminal deoxynucleotidyl transferase of the present disclosure. Disclosed herein include embodiments of host cells comprising a polynucleotide encoding the recombinant terminal deoxynucleotidyl transferase (TdT) of the present disclosure. Disclosed herein are embodiments of host cells comprising an expression vector comprising a polynucleotide encoding the recombinant terminal deoxynucleotidyl transferase of the present disclosure.

[0028] Disclosed herein include kits. In some embodiments, the kits comprise a recombinant terminal deoxynucleotidyl transferase (TdT) of the disclosure and instructions for using the recombinant terminal deoxynucleotidyl transferase. In some embodiments, the kits comprise a polynucleotide encoding a recombinant terminal deoxynucleotidyl transferase of the disclosure and instructions for using the polynucleotide and / or recombinant terminal deoxynucleotidyl transferase. In some embodiments, the kits comprise an expression vector comprising a polynucleotide encoding a recombinant terminal deoxynucleotidyl transferase of the disclosure and instructions for using the expression vector, polynucleotide, and / or recombinant terminal deoxynucleotidyl transferase. In some embodiments, the kit comprises a host cell comprising a recombinant terminal deoxynucleotidyl transferase of the present disclosure, a polynucleotide encoding the recombinant terminal deoxynucleotidyl transferase, an expression vector comprising a polynucleotide encoding the recombinant terminal deoxynucleotidyl transferase, or a combination thereof, and instructions for using the recombinant terminal deoxynucleotidyl transferase, the polynucleotide, the expression vector, the host cell, or a combination thereof.

[0029] The details of one or more implementations of the subject matter described herein are set forth in the accompanying drawings and the description below. Other features, aspects, and advantages will become apparent from the description, drawings, and claims. Neither this summary nor the following detailed description is intended to define or limit the scope of the inventive subject matter. [Brief explanation of the drawings]

[0030] [Figure 1]

[0023] Figure 1 shows a non-limiting exemplary sequence alignment of SUMO-TdT (SEQ ID NO: 14) with amino acids 139-520 (SEQ ID NO: 1) of Bos taurus TdT (SEQ ID NO: 12). SUMO-TdT refers to recombinant TdT that contains amino acids 139-520 of Bos taurus TdT at amino acid positions 123-504 and an N-terminal SUMO tag (SEQ ID NO: 13) at amino acid positions 22-119. The positions of substitution mutations in SUMO-TdT (and its TdT variants) identified herein and the corresponding positions in Bos taurus TdT are highlighted. [Figure 2] Figure 1 shows a non-limiting, exemplary schematic diagram of a FRET reaction for detecting the incorporation of Cy5-dCTP into an oligo by FAM dye-activated TdT, two bases away from the 3'-OH (2bA FAM). In the first round of screening, SUMO-TdT (recombinant bovine TdT lacking the first 138 amino acids of bovine TdT and containing an N-terminal SUMO tag to improve solubility and expression) was used as the parent template, and mutants TdT1-1 and TdT1-2 were identified as thermostable. In the second round of screening, TdT1-2 was used as the parent template and subjected to a 1-minute heat treatment at 50°C, resulting in the identification of four thermostable mutants (TdT2-1, TdT2-2, TdT2-3, and TdT2-4). In the third round of screening, TdT1-1 and TdT1-3 were used as parental templates, with different combinations of mutations found in TdT2-1, TdT2-2, TdT2-3 and TdT2-4, identifying TdT3-2 as thermostable. [Figure 3A] Establishment of a FRET assay for the detection of TdT activity. A non-limiting exemplary schematic diagram of a FRET reaction for detecting the incorporation of Cy5-dCTP into an oligo by activated TdT with FAM dye on a nucleotide two bases away from the 3'-OH (2bA FAM). [Figure 3B] Figure 3B shows the establishment of a FRET assay for the detection of TdT activity. Figure 3C shows a preliminary test of the schematic in Figure 3A to detect purified TdT activity corresponding to the increase in Cy5 emission at 670 nm with a FAM excitation wavelength of 450 nm. [Figure 3C]Figure 1 shows the establishment of a FRET assay for detecting TdT activity. This is a non-limiting, exemplary schematic diagram showing that endogenous nucleases in cell lysates degrade oligosubstrates at high concentrations. Dilution of the lysate disproportionately reduces nuclease activity relative to TdT, allowing detection of TdT activity. The scissors symbol indicates the nuclease. The star symbol indicates active TdT. [Figure 3D] Establishment of a FRET assay for the detection of TdT activity. Gel images showing various dilutions of TdT-expressing cell lysate in TdT buffer incubated with 5' FAM-tagged oligos (FAM20) and dCTP. The higher bands indicating TdT activity (boxed) are only seen at 50- and 500-fold dilutions; lower dilutions result in degradation by endogenous nucleases (below the blue line). [Figure 3E] Figure 1 shows the establishment of a FRET assay for the detection of TdT activity. Figure 2 shows non-limiting exemplary plots showing the results of a 96-well plate FRET assay using SUMO-TdT and empty plasmids using 50-fold diluted cell lysates. The higher FRET signal at 670 nm indicates expression of SUMO-TdT (red line) but not empty plasmid (blue line), suggesting that this assay can be used to screen for active TdT. [Figure 4A] Figure 1 shows emission wavelength scans of different FAM-tagged oligos with TAMRA-dCTP excited at 450 nm. Figure 2 shows non-limiting exemplary plots of emission wavelength scans of Oligo 1bA FAM with TAMRA-dCTP in the presence or absence of TdT. The decrease in FAM emission at 520 nm is low, suggesting inefficient energy transfer to the TAMRA dye. [Figure 4B] Figure 1 shows emission wavelength scans of different FAM-tagged oligos with TAMRA-dCTP excited at 450 nm. Figure 2 shows non-limiting exemplary plots of emission wavelength scans of oligo 2bA FAM with TAMRA-dCTP in the presence or absence of TdT. Upon incubation with TdT, the decrease in emission signal from FAM and the increase in emission signal from TAMRA appear to be the best of the four oligos investigated. [Figure 4C] 4A and 4B show emission wavelength scans of different FAM-tagged oligos with TAMRA-dCTP excited at 450 nm. Figure 4B shows non-limiting exemplary plots of emission wavelength scans of oligo 5bA FAM with TAMRA-dCTP in the presence or absence of TdT. The decrease in the emission signal of FAM and the increase in the emission signal of TAMRA in the presence of TdT are lower than those shown in Figure 4B. [Figure 4D]

[0033] Figure 1 shows emission wavelength scans of different FAM-tagged oligos with TAMRA-dCTP excited at 450 nm. Figure 2 shows non-limiting exemplary plots of emission wavelength scans of oligo 10bA FAM with TAMRA-dCTP in the presence or absence of TdT. There was no difference in the emission signals of FAM and TAMRA with or without TdT. [Figure 5A] Figure 1 shows emission wavelength scans of different FRET pairs. Figure 2 shows non-limiting exemplary plots of emission wavelength scans of the 2bA FAM oligo and TAMRA-dCTP in the presence or absence of TdT. Emission wavelengths were scanned from 480 nm to 700 nm with excitation at 450 nm. The 2bA oligo did not show a strong signal at 575 nm, which corresponds to the emission of the TAMRA acceptor dye. [Figure 5B] Figure 1 shows emission wavelength scans of different FRET pairs. Figure 1 shows non-limiting exemplary plots of emission wavelength scans of 2bA FAM oligo and Cy5-dCTP in the presence or absence of TdT. Emission wavelengths were scanned from 480 nm to 700 nm with excitation at 450 nm. In the presence of TdT, there was an increase in signal at 670 nm. This suggests successful detection of active TdT using the FAM and Cy5 FRET pair. [Figure 5C] Figure 5B shows emission wavelength scans of different FRET pairs. Figure 5C shows non-limiting exemplary plots of emission wavelength scans of 2bA FAM oligo and Cy3-dCTP in the presence or absence of TdT. Emission wavelengths were scanned from 530 nm to 700 nm with excitation at 500 nm. An oligo with Cy3 dye labeled on the second base from the 3'-OH was incubated with Cy5-dCTP and TdT. Compared to Figure 5B, lower signal was obtained at 670 nm. [Figure 6] 1 is a schematic diagram showing a non-limiting exemplary methodology for TdT thermostability screening. Two serial dilution steps (10-fold and 5-fold) result in a net 50-fold diluted lysate. [Figure 7A] Figure 1 shows FRET readouts of TdT variants across three rounds of thermostability screening. Figure 2 shows non-limiting exemplary histograms comparing the FRET activity of SUMO-TdT and variants TdT1-1 and TdT1-2. Unheated refers to cell lysates not heat-treated prior to incorporation into the reaction, while heated refers to cell lysates heated to 47°C for 1 minute prior to the reaction. FRET readouts for SUMO-TdT, TdT1-1, and TdT1-2 were recorded between 15 and 30 minutes of the reaction time course, and the average values ​​are displayed in the histograms above. Analysis was performed using a Student's T test with unequal variances, which indicates that the average FRET readouts for SUMO-TdT, TdT1-1, and TdT1-2 are significantly different. The average activity ratio of TdT1-1 and TdT1-2 was calculated by dividing the average FRET readout with heating by the average FRET readout without heating. TdT1-1 and TdT1-2 retained higher activity after heat treatment, both significantly higher than SUMO-TdT. [Figure 7B] FRET readout of TdT variants across three rounds of thermostability screening. Non-limiting exemplary histograms showing the second round of screening, heat treatment at 50° C. for 1 minute, identified variants TdT2-1, TdT2-2, TdT2-3, and TdT2-4 with significantly higher FRET values ​​and activity ratios than the parent TdT1-1. [Figure 7C] FRET readout of TdT variants across three rounds of thermostability screening. Non-limiting exemplary histograms showing the third round of screening using the parental template TdT1-1, where TdT3-1 was identified as having a significantly higher FRET readout and activity ratio than TdT1-1 after heat treatment at 55°C. [Figure 7D]FRET readout of TdT variants across three rounds of thermostability screening. Non-limiting exemplary histogram showing the third round of screening using parental template TdT1-3 (a hybrid of TdT1-1 and TdT1-2), where TdT3-2 had a significantly higher FRET readout and retained a significantly higher percentage of activity than TdT1-3 after 1 minute of heat treatment at 58°C. [Figure 8A] Figure 1 shows the purification of SUMO-TdT and TdT3-2 and verification of nuclease contamination in purified TdT. Figure 2 shows non-limiting exemplary gel images showing the purification of SUMO-TdT and TdT3-2. The yields of purified SUMO-TdT and TdT3-2 were 8.5 mg / L and 14.1 mg / L culture, respectively. SDS-PAGE gel of 1 μg of each dialysis-purified SUMO-TdT and TdT3-2. The expected size of SUMO-TdT is 57.9 kD, and that of TdT3-2 is 57.7 kD. A major band of approximately 58 kD ladder was observed for both SUMO-TdT and TdT3-2, corresponding to the respective proteins. The positions of the major bands indicate successful purification of SUMO-TdT and TdT3-2 by nickel-IMAC and Q Sepharose IEX chromatography. [Figure 8B] Figure 1 shows the purification of SUMO-TdT and TdT3-2 and verification of nuclease contamination in purified TdT. Figure 2 shows non-limiting, exemplary gel images showing a nuclease test to examine the presence of nuclease contamination in purified SUMO-TdT and TdT3-2. A 35-base single-stranded oligo substrate was incubated with SUMO-TdT and TdT3-2 separately. The samples were visualized on a TBE-urea gel. Observed bands smaller than 35 bases suggest the presence of nucleases in the protein sample that degrade the single-stranded oligo substrate. Lane 3 is a control to demonstrate the expected observation of degraded DNA products in the presence of nucleases. Comparing lanes 3, 4, and 5, the bands observed in lanes 4 and 5 are much smaller, suggesting the presence of minimal nucleases in the purified SUMO-TdT and TdT3-2. [Figure 9A]

[0023] Figure 1 shows Tm measurements of purified SUMO-TdT and TdT3-2. Figure 1 shows non-limiting exemplary plots of differential scanning calorimetry (DSC) of purified SUMO-TdT and TdT3-2. The peaks in the graph correspond to the protein transition midpoints (Tm or melting temperatures), where 50% of the protein is unfolded. The average Tm for SUMO-TdT was 40.2°C, and for TdT3-2 it was 50.7°C. [Figure 9B] Figure 1 shows Tm measurements of purified SUMO-TdT and TdT3-2. Figure 1 shows non-limiting exemplary plots of differential scanning fluorimetry (DSF) of purified SUMO-TdT and TdT3-2. The peaks observed in the graph of the negative derivative of F350nm / F330nm versus temperature correspond to the Tm of the protein. The average Tm for SUMO-TdT was 43.5°C, and for TdT3-2 it was 53.1°C. [Figure 9C] Figure 1 shows Tm measurements of purified SUMO-TdT and TdT3-2. Figure 2 shows non-limiting exemplary plots of the results of SYPRO Orange thermal shift assays of purified SUMO-TdT and TdT3-2. SYPRO Orange dye binds nonspecifically to hydrophobic regions of unfolded proteins. The peaks observed in the graph of -dRFU / temperature versus temperature correspond to the Tm of SUMO-TdT and TdT3-2. The average Tm for SUMO-TdT was 41.5°C, and for TdT3-2 it was 51.5°C. [Figure 9D] Figure 1 shows Tm measurements of purified SUMO-TdT and TdT3-2. This is a non-limiting exemplary plot of the first derivative of CD ellipticity at 222 nm as a function of temperature. Peaks observed in the graph correspond to the Tm of the proteins. The average Tm for SUMO-TdT was 45.7°C, and for TdT3-2 it was 52.5°C. [Figure 10A] 10A-10C are non-limiting exemplary gel images showing the observed activity of commercially available (NEB) TdT, SUMO-TdT, and TdT3-2 at various temperatures. All TdTs (FIG. 10A) were active at 25° C. [Figure 10B]10A-10C are non-limiting exemplary gel images showing the observed activity of commercially available (NEB) TdT, SUMO-TdT, and TdT3-2 at various temperatures. All TdTs were active at 36° C. (FIG. 10B). [Figure 10C] 1 shows non-limiting exemplary gel images showing the observed activity of commercially available (NEB) TdT, SUMO-TdT, and TdT3-2 at various temperatures. As observed from the higher-sized band DNA product, TdT3-2 was able to incorporate dCTP into 5' FAM-labeled oligos for 20 minutes at 47°C, while NEB TdT and SUMO-TdT denatured within 5 minutes. [Figure 10D] 1 is a non-limiting exemplary gel image showing the observation of commercial (NEB) TdT, SUMO-TdT, and TdT3-2 activity at various temperatures. It shows that only TdT3-2 was active at 58°C for less than 5 minutes. NEB TdT and SUMO-TdT were denatured. [Figure 11] 1 is a non-limiting exemplary plot examining the activity of commercially available TdT (NEB TdT) and TdT3-2 on blunt-ended ds DNA substrates. TdT3-2 was able to incorporate greater amounts of ddCTP into blunt-ended ds DNA substrates at both 37°C and 50°C. The percentage increased at 50°C. At 37°C for 4 minutes, the percentage of primers with ddCTP incorporated by NEB TdT was approximately 13.4%, while TdT3-2 was 55.7%. Over the same period at 50°C, the rate of ddCTP incorporation into primers by NEB TdT was approximately 8%, while TdT3-2 was approximately 89.9%. [Figure 12] 1 is a non-limiting exemplary gel graph showing ddCTP incorporation into FAM-tagged oligos with NEB TdT and TdT3-2. Band intensities were analyzed using Bio-Rad Image Lab software and plotted using GraphPad. TdT3-2 incorporated more ddCTP than NEB TdT, as observed by the higher intensity +1 band. At a higher temperature of 50°C, TdT3-2 incorporated more ddCTP than at 37°C, as observed by the increasing proportion of +1 bands at 50°C. [Figure 13] Figure 1 shows data on the binding affinity of SUMO-TdT and TdT3-2 to oligo substrates via fluorescence polarization. Varying amounts of SUMO-TdT and TdT3-2 were incubated with 5 nM of 5'-FAM-labeled oligo for 10 min at 37 °C before reading the polarization on a microplate reader. [Figure 14] 1 is a non-limiting, exemplary image showing the predicted protein structure of TdT3-2 using the Phyre2 web portal. Amino acid residues in TdT3-2 that differ from SUMO-TdT are highlighted in darker shading and labeled as indicated. Residues highlighted in lighter shading (338D, 340D, and 428D) correspond to residues that bind divalent metal ions during the incorporation reaction. Residues that bind DNA are on the highlighted alpha helix (residues 253-257). Loop 1 of TdT3-2 is highlighted (residues 376-394). DETAILED DESCRIPTION OF THE INVENTION

[0031] In the following detailed description, reference is made to the accompanying drawings, which form a part of this specification. In the drawings, like symbols typically identify like elements unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not intended to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that aspects of the present disclosure, as generally described herein and illustrated in the drawings, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are expressly contemplated herein and make part of this disclosure.

[0032] All patents, published patent applications, other publications, and sequences from GenBank and other databases referenced herein are hereby incorporated by reference in their entirety for relevant art.

[0033] Sequencing by synthesis has enabled the discovery of vast amounts of information about the underlying source code of life. To fully benefit from this information, it is necessary to create entirely synthetic genes and genomes to test whether the understanding derived from sequencing can be used to predictably create new systems. For example, the construction of synthetic organisms using genomes constructed using chemically synthesized oligos has been demonstrated. Similarly, efforts to create synthetic yeast and E. coli with only 57 codons instead of 64, or even to recreate extinct organisms (References 6 and 7), represent the next advance in bioengineering.

[0034] However, this effort is hindered by the fact that current oligonucleotide and gene synthesis methods rely on chemical synthesis using phosphoramidite chemistry. This method imposes many limitations, such as a maximum size of less than 200 bp, due to deletions and side reactions, and the use and generation of environmentally harmful organic chemicals and waste products. As a result, the cost of genome-scale construction runs into the millions of dollars, which is a major obstacle.

[0035] Wild-type terminal deoxynucleotidyl transferase is highly unstable and not optimized for enzymatic oligonucleotide and gene synthesis using stepwise incorporation and deprotection of a single 3'-blocked nucleotide into a growing ssDNA strand. An engineered TdT that can accept 3'-blocked nucleotides and has increased activity and robustness is needed.

[0036] Terminal deoxynucleotidyl transferase (TdT) catalyzes the template-free incorporation of any nucleotide into single-stranded DNA. TdT is widely used in biotechnology and clinical applications. One possible use is the synthesis of long de novo DNA molecules by TdT-mediated iterative incorporation of 3' reversibly blocked nucleotides followed by subsequent deblocking. However, wild-type (WT) TdT is not optimized for the incorporation of 3'-modified nucleotides, and TdT engineering is hindered by the fact that TdT is only marginally stable and present only in mesophilic organisms. Thermostable TdT variants could serve as scaffolds for subsequent evolution to enable efficient incorporation of 3'-modified nucleotides. Because evolution to incorporate bulky modified nucleotides generally results in decreased stability, thermostable variants would be a good starting point for such efforts. Furthermore, thermostable TdT would be useful in situations where DNA secondary structures inhibit WT activity, as higher temperatures can be used to melt dsDNA. The assay described in this disclosure was developed to identify thermostable TdT variants. After screening approximately 10,000 TdT mutants, we identified a variant, designated TdT3-2, that was 10°C more thermostable than SUMO-TdT and retained the catalytic properties of the WT enzyme. Any of the recombinant TdTs disclosed herein can be used as a scaffold for evolving TdTs capable of incorporating 3'-blocking nucleotides into single-stranded DNA.

[0037] Several embodiments of recombinant terminal deoxynucleotidyl transferase (TdT) are provided herein. In some embodiments, the recombinant TdT comprises an amino acid sequence at least 80% identical to SEQ ID NO: 1, and the recombinant TdT comprises one or more amino acid substitution mutations at one or more positions functionally equivalent to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 of Bos taurus TdT of SEQ ID NO: 12. Disclosed herein are embodiments of polynucleotides encoding the recombinant terminal deoxynucleotidyl transferase. Disclosed herein are embodiments of expression vectors comprising a polynucleotide encoding the recombinant terminal deoxynucleotidyl transferase. Disclosed herein are embodiments of host cells comprising the recombinant terminal deoxynucleotidyl transferase. Disclosed herein are embodiments of host cells comprising a polynucleotide encoding the recombinant terminal deoxynucleotidyl transferase. Disclosed herein include embodiments of host cells that contain expression vectors that include polynucleotides that encode recombinant terminal deoxynucleotidyls.

[0038] definition Unless otherwise defined, technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. See, for example, Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor, NY 1989). For purposes of this disclosure, the following terms are defined below.

[0039] The terms "identical" or "percent identity" in the context of two or more nucleic acid or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same when compared and aligned for maximum correspondence, as determined using a sequence comparison algorithm or by visual inspection.

[0040] The phrase "substantially identical" in the context of two nucleic acids or polypeptides (e.g., DNA encoding a protein, or the amino acid sequence of a protein) refers to two or more sequences or subsequences that, when compared and aligned for maximum correspondence, have at least about 60%, about 80%, about 90-95%, about 98%, or about 99% or more nucleotide or amino acid residue identity, as determined using a sequence comparison algorithm or by visual inspection. Such "substantially identical" sequences are typically considered to be "homologous" without reference to actual ancestry. "Substantial identity" can exist over a region of the sequences that is at least about 50 residues in length, more preferably over a region of at least about 100 residues, and most preferably, the sequences are substantially identical over a region of at least about 150 residues, or over the entire length of the two sequences being compared.

[0041] Proteins and / or protein sequences are "homologous" if they are derived, naturally or artificially, from a common ancestral protein or protein sequence. Similarly, nucleic acids and / or nucleic acid sequences are homologous if they are derived, naturally or artificially, from a common ancestral nucleic acid or nucleic acid sequence. Homology is generally inferred from sequence similarity between two or more nucleic acids or proteins (or sequences thereof). The exact percentage of similarity between sequences useful for establishing homology varies depending on the nucleic acid and protein in question, but sequence similarity of as little as 25% over 50, 100, 150 or more residues is routinely used to establish homology. Higher levels of sequence similarity, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% or more, can also be used to establish homology. Methods for determining sequence similarity percentages (e.g., BLASTP and BLASTN) are available.

[0042] For sequence comparison and homology determination, typically, one sequence serves as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated as necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence(s) relative to the reference sequence based on the designated program parameters.

[0043] Optimal alignment of sequences for comparison can be performed, for example, by local homology algorithms, homology alignment algorithms, search for similarity methods, computerized implementations of these algorithms, or by visual inspection. One example of an algorithm suitable for determining percent sequence identity and percent sequence similarity is the BLAST algorithm. Software for performing BLAST analysis is publicly available from the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in a query sequence that, when aligned with words of the same length in a database sequence, match or meet some positive threshold score T. T is referred to as the neighborhood word score threshold. These initial neighborhood word hits serve as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues, always >0) and N (penalty score for mismatching residues, always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of word hits in each direction is stopped when the cumulative alignment score falls by an amount X from its maximum achieved value, the cumulative score falls below 0 due to the accumulation of one or more negative-scoring residue alignments, or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM 62 scoring matrix.

[0044] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered to be similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid with the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[0045] Amino acids at "functionally equivalent" amino acid positions can have the same functional role in proteins (e.g., enzymes). Generally, functionally equivalent substitution mutations in two or more different proteins occur at homologous amino acid positions within the amino acid sequences of the proteins. Thus, as used herein, the term "functionally equivalent" also encompasses mutations that are "positionally equivalent" or "homologous" to a given mutation, regardless of whether the specific function of the mutant amino acid is known. Positionally equivalent or homologous amino acid residues in the amino acid sequences of two or more different proteins can be identified based on sequence alignment and / or molecular modeling.

[0046] Terminal deoxynucleotidyl transferase engineering Terminal deoxynucleotidyl transferase (TdT) was one of the first DNA polymerases discovered in mammals. TdT possesses template-independent activity. This activity can increase the diversity of the body's antibody repertoire. TdT can catalyze the addition of nucleotides to single-stranded DNA primers at least three nucleotides long in the presence of divalent metal ions. As a member of the X family of DNA polymerases, TdT is most similar to Pol μ, a template-dependent DNA polymerase. It shares 42% sequence similarity with Pol μ. Without being bound by any particular theory, the factor contributing to the difference in template dependence between TdT and Pol μ is thought to be the presence of a flexible loop (called Loop 1). Destabilization of Loop 1 in TdT has been shown to confer template-dependent activity. Switching Loop 1 between TdT and Pol μ may result in a partial change in DNA substrate preference.

[0047] Due to its unique activity, TdT has been used in clinical and biotechnology fields. One application of TdT is the TUNEL assay, in which DNA fragments from apoptotic cells are detected by TdT-catalyzed incorporation of fluorophore-labeled nucleotides into exposed DNA fragments. TdT can also be used in methods to detect small amounts of DNA and RNA for viral detection and diagnosis of genetic diseases.

[0048] Being a template-independent DNA polymerase, TdT is an ideal candidate for enzymatic DNA synthesis. Nucleotide-conjugated TdT has been used to enable single incorporation into DNA initiators. The potential of TdT for de novo DNA synthesis can also be exploited to enable data storage in DNA. DNA has the advantages of extremely high information density and low maintenance storage, allowing for data quality to be maintained at 10 per gram of DNA for hundreds of years. 15 ~10 20byte of data. TdT-mediated synthesis of homopolymer stretches and transitions between them has been used to encode digital data. However, these methods have several inherent drawbacks, such as complex synthesis processes or the generation of heterogeneous products. An alternative method for enzymatically generating arbitrary DNA sequences would be to use TdT to reversibly incorporate 3'-blocked nucleotides, such as 3'-O-blocked nucleotides, including 3'-O-amino-blocked nucleotides and 3'-O-azidomethyl-blocked nucleotides. User-friendly DNA synthesis machines using enzymatic synthesis methods have not yet been commercially available.

[0049] Wild-type (WT) TdT cannot incorporate bulky 3'-blocked nucleotides due to steric clashes. TdT can be engineered to accept reversibly blocked nucleotides for use in sequencing, such as sequencing by synthesis. However, engineering TdT to incorporate reversibly blocked nucleotides is difficult. This is due, in part, to the fact that TdT has a T of approximately 40°C. m This is because TdT is a mesophilic polymerase with limited stability. Mutations that confer desired activity can often be destabilizing. Therefore, to obtain a more stable initial construct for further manipulation, it is first necessary to improve the thermostability of TdT.

[0050] Furthermore, enzymatic DNA synthesis may need to overcome the formation of strong DNA secondary structures, such as hairpins, as the length of the DNA product increases. One possible solution is to increase the temperature of the enzymatic DNA synthesis process. However, the optimum temperature for WT TdT is approximately 37°C, and the unfolded T m is approximately 40 °C. Therefore, engineering a thermostable TdT helps minimize the formation of secondary DNA structures during de novo DNA synthesis by allowing synthesis at higher temperatures.

[0051] Disclosed herein are TdT variants (also referred to herein as TdT mutants and recombinant TdT) evolved to have improved thermostability. A fluorescence-based TdT activity assay was developed that can be performed on cell lysates after incubation at increasing temperatures (Figures 2 and 3). Second, expression and purification protocols were optimized to obtain adequate yields of WT and mutant enzymes. Third, the thermostability of the TdT variants was verified by DSC, DSF, SYPRO Orange thermal shift assays, and CD. Finally, kinetic characterization of a thermostabilized TdT variant, referred to herein as TdT3-2, was performed to demonstrate that the increased thermostability did not result in a sacrifice of enzyme activity.

[0052] Recombinant terminal deoxynucleotidyl transferase array Disclosed herein are embodiments of recombinant terminal deoxynucleotidyl transferase (TdT). In some embodiments, the recombinant TdT is derived from bovine or Bos taurus The amino acid sequence of the TdT fragment (e.g., SEQ ID NO: 12) may be at or near 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to TdT, or a fragment thereof (e.g., SEQ ID NO: 12). In some embodiments, the recombinant TdT is homologous to bovine or Bos taurus TdT (e.g., SEQ ID NO: 12) by at least, at least about, at most, or at most about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, The recombinant TdT may comprise an amino acid sequence that is at least 85% identical to SEQ ID NO: 1. The recombinant TdT may comprise an amino acid sequence that may be at least 90% identical to SEQ ID NO: 1. The recombinant TdT may comprise an amino acid sequence that may be at least 95% identical to SEQ ID NO: 1.

[0053] In some embodiments, the recombinant TdT comprises an amino acid sequence that has sequence identity above a sequence identity threshold to bovine or a fragment of Bos taurus TdT, such as amino acids 139-520 of Bos taurus TdT (e.g., SEQ ID NO: 1). FIG. 1 shows the sequence of amino acids 139-520 of Bos taurus TdT. For example, the recombinant terminal deoxynucleotidyl transferase (TdT) can comprise an amino acid sequence at least 80% identical to SEQ ID NO: 1. In some embodiments, the recombinant TdT comprises an amino acid sequence that has sequence identity above a sequence identity threshold to a variant of bovine or Bos taurus TdT, or a variant of a bovine or Bos taurus TdT fragment (e.g., SEQ ID NO: 11). For example, the recombinant TdT can comprise an amino acid sequence that can be at least 95% identical to SEQ ID NO: 11.

[0054] Substitution mutation The recombinant TdT can include one or more amino acid substitution mutations at one or more positions functionally equivalent to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 of Bos taurus TdT (e.g., SEQ ID NO: 12). Each amino acid substitution mutation can be an amino acid substitution mutation to alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0055] Each amino acid substitution mutation can be a substitution mutation for a nonpolar amino acid, a polar amino acid, a positively charged amino acid, a negatively charged amino acid, a hydrophobic amino acid, an aromatic amino acid, an aliphatic amino acid, a small amino acid, a hydrophilic amino acid, or a branched-chain amino acid. Nonpolar amino acids can be, for example, alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, or valine. Polar amino acids can be, for example, aspartic acid, glutamic acid, arginine, histidine, lysine, asparagine, glutamine, serine, threonine, or tyrosine. Polar amino acids can be, for example, acidic polar amino acids, basic polar amino acids, or nonacidic nonbasic polar amino acids. Basic polar amino acids or positively charged amino acids can be, for example, arginine, histidine, or lysine. Acidic amino acids or negatively charged amino acids can be, for example, aspartic acid or glutamic acid. Non-acidic, non-basic amino acids may be, for example, asparagine, glutamine, serine, threonine, or tyrosine. Hydrophobic amino acids may be, for example, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, or valine. Aromatic amino acids may be, for example, histidine, phenylalanine, tryptophan, or tyrosine. Aliphatic (non-aromatic) amino acids may be, for example, isoleucine, leucine, methionine, or valine. Small amino acids may be, for example, alanine, glycine, proline, or serine. Hydrophilic amino acids may be, for example, arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, serine, or threonine. Branched-chain amino acids may be, for example, isoleucine, leucine, or valine.

[0056] In some embodiments, the amino acid substitution mutation at a position functionally equivalent to Glu191 of Bos taurus TdT of SEQ ID NO: 12 includes a mutation to a nonpolar amino acid, a polar amino acid, a positively charged amino acid, a negatively charged amino acid, a hydrophobic amino acid, an aromatic amino acid, an aliphatic amino acid, a small amino acid, or a hydrophilic amino acid. The amino acid substitution mutation at a position functionally equivalent to Glu191 can include a mutation to a nonpolar amino acid, a hydrophobic amino acid, an aliphatic amino acid, or a branched-chain amino acid. The amino acid substitution mutation at a position functionally equivalent to Glu191 of Bos taurus TdT of SEQ ID NO: 12 can be an amino acid substitution mutation to alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine. The amino acid substitution mutation at a position functionally equivalent to Glu191 of Bos taurus TdT of SEQ ID NO: 12 can be an amino acid substitution mutation to alanine, glycine, isoleucine, leucine, methionine, or valine. The amino acid substitution mutation at a position functionally equivalent to Glu191 of Bos taurus TdT of SEQ ID NO: 12 can be Glu191Ala, Glu191Arg, Glu191Asn, Glu191Asp, Glu191Cys, Glu191Gln, Glu191Gly, Glu191His, Glu191Ile, Glu191Leu, Glu191Lys, Glu191Met, Glu191Phe, Glu191Pro, Glu191Ser, Glu191Thr, Glu191Trp, Glu191Tyr, or Glu191Val. The amino acid substitution mutation at a position functionally equivalent to Glu191 of Bos taurus TdT of SEQ ID NO: 12 can be Glu191Ala, Glu191Gly, Glu191Ile, Glu191Leu, Glu191Met, or Glu191Val. The amino acid substitution mutation at a position functionally equivalent to Glu191 of Bos taurus TdT of SEQ ID NO: 12 can be Glu191Val. The recombinant TdT can comprise an amino acid sequence having sequence identity to SEQ ID NO: 2 that exceeds a sequence identity threshold.

[0057] In some embodiments, the amino acid substitution mutation at a position functionally equivalent to Lys193 of Bos taurus TdT of SEQ ID NO: 12 comprises a mutation to a nonpolar amino acid, a polar amino acid, a positively charged amino acid, a negatively charged amino acid, a hydrophobic amino acid, an aromatic amino acid, an aliphatic amino acid, a small amino acid, or a hydrophilic amino acid. The amino acid substitution mutation at a position functionally equivalent to Lys193 of Bos taurus TdT of SEQ ID NO: 12 can comprise a mutation to a polar amino acid or a hydrophilic amino acid. The amino acid substitution mutation at a position functionally equivalent to Lys193 of Bos taurus TdT of SEQ ID NO: 12 can be an amino acid substitution mutation to alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine. The amino acid substitution mutation at a position functionally equivalent to Lys193 of Bos taurus TdT of SEQ ID NO: 12 can be an amino acid substitution mutation to asparagine, glutamine, serine, or threonine. The amino acid substitution mutation at a position functionally equivalent to Lys193 of Bos taurus TdT of SEQ ID NO: 12 can be Lys193Ala, Lys193Arg, Lys193Asn, Lys193Asp, Lys193Cys, Lys193Gln, Lys193Glu, Lys193Gly, Lys193His, Lys193Ile, Lys193Leu, Lys193Met, Lys193Phe, Lys193Pro, Lys193Ser, Lys193Thr, Lys193Trp, Lys193Tyr, or Lys193Val. The amino acid substitution mutation at a position functionally equivalent to Lys193 of Bos taurus TdT of SEQ ID NO: 12 can be Lys193Asn, Lys193Gln, Lys193Ser, or Lys193Thr. The amino acid substitution mutation at a position functionally equivalent to Lys193 of Bos taurus TdT of SEQ ID NO: 12 can be Lys193Asn. The recombinant TdT can comprise an amino acid sequence having sequence identity to SEQ ID NO: 3 above a sequence identity threshold.

[0058] In some embodiments, the amino acid substitution mutation at a position functionally equivalent to Glu194 of Bos taurus TdT of SEQ ID NO: 12 comprises a mutation to a nonpolar amino acid, a polar amino acid, a positively charged amino acid, a negatively charged amino acid, a hydrophobic amino acid, an aromatic amino acid, an aliphatic amino acid, a small amino acid, or a hydrophilic amino acid. The amino acid substitution mutation at a position functionally equivalent to Glu194 of Bos taurus TdT of SEQ ID NO: 12 can comprise a mutation to a nonpolar amino acid, a hydrophobic amino acid, an aliphatic amino acid, or a branched-chain amino acid. The amino acid substitution mutation at a position functionally equivalent to Glu194 of Bos taurus TdT of SEQ ID NO: 12 can be an amino acid substitution mutation to alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine. The amino acid substitution mutation at a position functionally equivalent to Glu194 of Bos taurus TdT of SEQ ID NO: 12 can be an amino acid substitution mutation to alanine, glycine, isoleucine, leucine, methionine, or valine. The amino acid substitution mutation at a position functionally equivalent to Glu194 of Bos taurus TdT of SEQ ID NO: 12 can be Glu194Ala, Glu194Arg, Glu194Asn, Glu194Asp, Glu194Cys, Glu194Gln, Glu194Gly, Glu194His, Glu194Ile, Glu194Leu, Glu194Lys, Glu194Met, Glu194Phe, Glu194Pro, Glu194Ser, Glu194Thr, Glu194Trp, Glu194Tyr, or Glu194Val. The amino acid substitution mutation at a position functionally equivalent to Glu194 of Bos taurus TdT of SEQ ID NO: 12 can be Glu194Ala, Glu194Gly, Glu194Ile, Glu194Leu, Glu194Met, or Glu194Val. The amino acid substitution mutation at a position functionally equivalent to Glu194 of Bos taurus TdT of SEQ ID NO: 12 can be Glu194Gly. The recombinant TdT can comprise an amino acid sequence having sequence identity to SEQ ID NO: 4 above a sequence identity threshold.

[0059] In some embodiments, the amino acid substitution mutation at a position functionally equivalent to Asp242 of Bos taurus TdT of SEQ ID NO: 12 comprises a mutation to a nonpolar amino acid, a polar amino acid, a positively charged amino acid, a negatively charged amino acid, a hydrophobic amino acid, an aromatic amino acid, an aliphatic amino acid, a small amino acid, or a hydrophilic amino acid. The amino acid substitution mutation at a position functionally equivalent to Asp242 of Bos taurus TdT of SEQ ID NO: 12 can comprise a mutation to a polar amino acid or an aromatic amino acid. The amino acid substitution mutation at a position functionally equivalent to Asp242 of Bos taurus TdT of SEQ ID NO: 12 can be an amino acid substitution mutation to alanine, arginine, asparagine, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine. The amino acid substitution mutation at a position functionally equivalent to Asp242 of Bos taurus TdT of SEQ ID NO: 12 can be an amino acid substitution mutation to asparagine, glutamine, phenylalanine, serine, threonine, tryptophan, or tyrosine. The amino acid substitution mutation at a position functionally equivalent to Asp242 of Bos taurus TdT of SEQ ID NO: 12 can be Asp242Ala, Asp242Arg, Asp242Asn, Asp242Cys, Asp242Gln, Asp242Glu, Asp242Gly, Asp242His, Asp242Ile, Asp242Leu, Asp242Lys, Asp242Met, Asp242Phe, Asp242Pro, Asp242Ser, Asp242Thr, Asp242Trp, Asp242Tyr, or Asp242Val. The amino acid substitution mutation at a position functionally equivalent to Asp242 of Bos taurus TdT of SEQ ID NO: 12 can be Asp242Asn, Asp242Gln, Asp242Phe, Asp242Ser, Asp242Thr, Asp242Trp, or Asp242Tyr. The amino acid substitution mutation at a position functionally equivalent to Asp242 of Bos taurus TdT of SEQ ID NO: 12 can be Asp242Tyr.The recombinant TdT can comprise an amino acid sequence having sequence identity to SEQ ID NO:5 above a threshold sequence identity.

[0060] In some embodiments, the amino acid substitution mutation at a position functionally equivalent to Lys287 of Bos taurus TdT of SEQ ID NO: 12 comprises a mutation to a nonpolar amino acid, a polar amino acid, a positively charged amino acid, a negatively charged amino acid, a hydrophobic amino acid, an aromatic amino acid, an aliphatic amino acid, a small amino acid, or a hydrophilic amino acid. The amino acid substitution mutation at a position functionally equivalent to Lys287 of Bos taurus TdT of SEQ ID NO: 12 can comprise a mutation to a polar amino acid, a negatively charged amino acid, or a hydrophilic amino acid. The amino acid substitution mutation at a position functionally equivalent to Lys287 of Bos taurus TdT of SEQ ID NO: 12 can be an amino acid substitution mutation to alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine. The amino acid substitution mutation at a position functionally equivalent to Lys287 of Bos taurus TdT of SEQ ID NO: 12 can be an amino acid substitution mutation to aspartic acid or glutamic acid. The amino acid substitution mutation at a position functionally equivalent to Lys287 of Bos taurus TdT of SEQ ID NO: 12 can be Lys287Ala, Lys287Arg, Lys287Asn, Lys287Asp, Lys287Cys, Lys287Gln, Lys287Glu, Lys287Gly, Lys287His, Lys287Ile, Lys287Leu, Lys287Met, Lys287Phe, Lys287Pro, Lys287Ser, Lys287Thr, Lys287Trp, Lys287Tyr, or Lys287Val. The amino acid substitution mutation at a position functionally equivalent to Lys287 of Bos taurus TdT of SEQ ID NO: 12 can be Lys287Asp or Lys287Glu. The amino acid substitution mutation at a position functionally equivalent to Lys287 of Bos taurus TdT of SEQ ID NO: 12 can be Lys287Glu. The recombinant TdT can comprise an amino acid sequence having sequence identity to SEQ ID NO: 6 that exceeds a sequence identity threshold.

[0061] In some embodiments, the amino acid substitution mutation at a position functionally equivalent to Phe296 of Bos taurus TdT of SEQ ID NO: 12 comprises a mutation to a nonpolar amino acid, a polar amino acid, a positively charged amino acid, a negatively charged amino acid, a hydrophobic amino acid, an aromatic amino acid, an aliphatic amino acid, a small amino acid, or a hydrophilic amino acid. The amino acid substitution mutation at a position functionally equivalent to Phe296 of Bos taurus TdT of SEQ ID NO: 12 can comprise a mutation to a nonpolar amino acid, a hydrophobic amino acid, an aliphatic amino acid, or a branched-chain amino acid. The amino acid substitution mutation at a position functionally equivalent to Phe296 of Bos taurus TdT of SEQ ID NO: 12 can be an amino acid substitution mutation to alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, proline, serine, threonine, tryptophan, tyrosine, or valine. The amino acid substitution mutation at a position functionally equivalent to Phe296 of Bos taurus TdT of SEQ ID NO: 12 can be an amino acid substitution mutation to alanine, glycine, isoleucine, leucine, methionine, or valine. The amino acid substitution mutation at a position functionally equivalent to Phe296 of Bos taurus TdT of SEQ ID NO: 12 can be Phe296Ala, Phe296Arg, Phe296Asn, Phe296Asp, Phe296Cys, Phe296Gln, Phe296Glu, Phe296Gly, Phe296His, Phe296Ile, Phe296Leu, Phe296Lys, Phe296Met, Phe296Pro, Phe296Ser, Phe296Thr, Phe296Trp, Phe296Tyr, or Phe296Val. The amino acid substitution mutation at a position functionally equivalent to Phe296 of Bos taurus TdT of SEQ ID NO: 12 can be Phe296Ala, Phe296Gly, Phe296Ile, Phe296Leu, Phe296Met, or Phe296Val. The amino acid substitution mutation at a position functionally equivalent to Phe296 of Bos taurus TdT of SEQ ID NO: 12 can be Phe296Leu. The recombinant TdT can comprise an amino acid sequence having sequence identity to SEQ ID NO: 7 above a sequence identity threshold.

[0062] In some embodiments, the amino acid substitution mutation at a position functionally equivalent to Met299 of Bos taurus TdT of SEQ ID NO: 12 includes a mutation to a polar amino acid, a positively charged amino acid, or a hydrophilic amino acid, or a mutation to a nonpolar amino acid, a polar amino acid, a positively charged amino acid, a negatively charged amino acid, a hydrophobic amino acid, an aromatic amino acid, an aliphatic amino acid, a small amino acid, or a hydrophilic amino acid. The amino acid substitution mutation at a position functionally equivalent to Met299 of Bos taurus TdT of SEQ ID NO: 12 can include a mutation to a polar amino acid, a positively charged amino acid, or a hydrophilic amino acid. The amino acid substitution mutation at a position functionally equivalent to Met299 of Bos taurus TdT of SEQ ID NO: 12 can be an amino acid substitution mutation to alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine. The amino acid substitution mutation at a position functionally equivalent to Met299 of Bos taurus TdT of SEQ ID NO: 12 can be an amino acid substitution mutation to arginine, arginine, histidine, or lysine. The amino acid substitution mutation at a position functionally equivalent to Met299 of Bos taurus TdT of SEQ ID NO: 12 can be Met299Ala, Met299Arg, Met299Asn, Met299Asp, Met299Cys, Met299Gln, Met299Glu, Met299Gly, Met299His, Met299Ile, Met299Leu, Met299Lys, Met299Phe, Met299Pro, Met299Ser, Met299Thr, Met299Trp, Met299Tyr, or Met299Val. The amino acid substitution mutation at a position functionally equivalent to Met299 of Bos taurus TdT of SEQ ID NO: 12 can be Met299Arg, Met299His, or Met299Lys. The amino acid substitution mutation at a position functionally equivalent to Met299 of Bos taurus TdT of SEQ ID NO: 12 can be Met299Lys. The recombinant TdT can comprise an amino acid sequence having sequence identity to SEQ ID NO: 8 that exceeds a sequence identity threshold.

[0063] In some embodiments, the amino acid substitution mutation at a position functionally equivalent to Thr342 of Bos taurus TdT of SEQ ID NO: 12 includes a mutation to a polar amino acid, an aliphatic amino acid, or a hydrophilic amino acid, or a mutation to a nonpolar amino acid, a polar amino acid, a positively charged amino acid, a negatively charged amino acid, a hydrophobic amino acid, an aromatic amino acid, an aliphatic amino acid, a small amino acid, or a hydrophilic amino acid. The amino acid substitution mutation at a position functionally equivalent to Thr342 of Bos taurus TdT of SEQ ID NO: 12 can include a mutation to a polar amino acid, an aliphatic amino acid, or a hydrophilic amino acid. The amino acid substitution mutation at a position functionally equivalent to Thr342 of Bos taurus TdT of SEQ ID NO: 12 can be an amino acid substitution mutation to alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, tryptophan, tyrosine, or valine. The amino acid substitution mutation at a position functionally equivalent to Thr342 of Bos taurus TdT of SEQ ID NO: 12 can be an amino acid substitution mutation to asparagine, cystine, glutamine, proline, or serine. The amino acid substitution mutation at a position functionally equivalent to Thr342 of Bos taurus TdT of SEQ ID NO: 12 can be Thr342Ala, Thr342Arg, Thr342Asn, Thr342Asp, Thr342Cys, Thr342Gln, Thr342Glu, Thr342Gly, Thr342His, Thr342Ile, Thr342Leu, Thr342Lys, Thr342Met, Thr342Phe, Thr342Pro, Thr342Ser, Thr342Trp, Thr342Tyr, or Thr342Val. The amino acid substitution mutation at a position functionally equivalent to Thr342 of Bos taurus TdT of SEQ ID NO: 12 can be Thr342Asn, Thr342Cys, Thr342Gln, Thr342Pro, or Thr342Ser. The amino acid substitution mutation at a position functionally equivalent to Thr342 of Bos taurus TdT of SEQ ID NO: 12 can be Thr342Ser.The recombinant TdT can comprise an amino acid sequence having sequence identity to SEQ ID NO:9 above a threshold sequence identity.

[0064] In some embodiments, the amino acid substitution mutation at a position functionally equivalent to His421 of Bos taurus TdT of SEQ ID NO: 12 includes a mutation to a polar amino acid, an aliphatic amino acid, or a hydrophilic amino acid, or a mutation to a nonpolar amino acid, a polar amino acid, a positively charged amino acid, a negatively charged amino acid, a hydrophobic amino acid, an aromatic amino acid, an aliphatic amino acid, a small amino acid, or a hydrophilic amino acid. The amino acid substitution mutation at a position functionally equivalent to His421 of Bos taurus TdT of SEQ ID NO: 12 can include a mutation to a polar amino acid, an aliphatic amino acid, or a hydrophilic amino acid. The amino acid substitution mutation at a position functionally equivalent to His421 of Bos taurus TdT of SEQ ID NO: 12 can be an amino acid substitution mutation to alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine. The amino acid substitution mutation at a position functionally equivalent to His421 of Bos taurus TdT of SEQ ID NO: 12 can be an amino acid substitution mutation to asparagine, cystine, glutamine, proline, serine, or threonine. The amino acid substitution mutation at a position functionally equivalent to His421 of Bos taurus TdT of SEQ ID NO: 12 can be His421Ala, His421Arg, His421Asn, His421Asp, His421Cys, His421Gln, His421Glu, His421Gly, His421Ile, His421Leu, His421Lys, His421Met, His421Phe, His421Pro, His421Ser, His421Thr, His421Trp, His421Tyr, or His421Val. The amino acid substitution mutation at a position functionally equivalent to His421 of Bos taurus TdT of SEQ ID NO: 12 can be His421Asn, His421Cys, His421Gln, His421Pro, His421Ser, or His421Thr. The amino acid substitution mutation at a position functionally equivalent to His421 of Bos taurus TdT of SEQ ID NO: 12 can be His421Pro.The recombinant TdT can comprise an amino acid sequence having sequence identity to SEQ ID NO:10 above a threshold sequence identity.

[0065] In some embodiments, the recombinant TdT comprises two or more amino acid substitution mutations at two or more positions functionally equivalent to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 of Bos taurus TdT of SEQ ID NO: 12. The recombinant TdT can comprise three or more amino acid substitution mutations at three or more positions functionally equivalent to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 of Bos taurus TdT of SEQ ID NO: 12. The recombinant TdT can include four or more amino acid substitution mutations at four or more positions functionally equivalent to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 of Bos taurus TdT of SEQ ID NO: 12. The recombinant TdT can include five or more amino acid substitution mutations at five or more positions functionally equivalent to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 of Bos taurus TdT of SEQ ID NO: 12. The recombinant TdT can include six or more amino acid substitution mutations at six or more positions functionally equivalent to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 of Bos taurus TdT of SEQ ID NO: 12. The recombinant TdT can include seven or more amino acid substitution mutations at seven or more positions functionally equivalent to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 of Bos taurus TdT of SEQ ID NO: 12. The recombinant TdT can include eight or more amino acid substitution mutations at eight or more positions functionally equivalent to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 of Bos taurus TdT of SEQ ID NO:12.

[0066] In some embodiments, the two or more amino acid substitution mutations at two or more positions functionally equivalent to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 of Bos taurus TdT of SEQ ID NO: 12 include two or more of Glu191Val, Lys193Asn, Glu194Gly, Asp242Tyr, Lys287Glu, Phe296Leu, Met299Lys, Thr342Ser, and His421Pro, respectively. The three or more amino acid substitution mutations at three or more positions functionally equivalent to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 of Bos taurus TdT of SEQ ID NO: 12 can include three or more of Glu191Val, Lys193Asn, Glu194Gly, Asp242Tyr, Lys287Glu, Phe296Leu, Met299Lys, Thr342Ser, and His421Pro, respectively. The four or more amino acid substitution mutations at four or more positions functionally equivalent to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 of Bos taurus TdT of SEQ ID NO: 12 can include four or more of Glu191Val, Lys193Asn, Glu194Gly, Asp242Tyr, Lys287Glu, Phe296Leu, Met299Lys, Thr342Ser, and His421Pro, respectively. The five or more amino acid substitution mutations at five or more positions functionally equivalent to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 of Bos taurus TdT of SEQ ID NO: 12 can include five or more of Glu191Val, Lys193Asn, Glu194Gly, Asp242Tyr, Lys287Glu, Phe296Leu, Met299Lys, Thr342Ser, and His421Pro, respectively.The six or more amino acid substitution mutations at six or more positions functionally equivalent to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 of Bos taurus TdT of SEQ ID NO: 12 can include six or more of Glu191Val, Lys193Asn, Glu194Gly, Asp242Tyr, Lys287Glu, Phe296Leu, Met299Lys, Thr342Ser, and His421Pro, respectively. The seven or more amino acid substitution mutations at seven or more positions functionally equivalent to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 of Bos taurus TdT of SEQ ID NO: 12 can include seven or more of Glu191Val, Lys193Asn, Glu194Gly, Asp242Tyr, Lys287Glu, Phe296Leu, Met299Lys, Thr342Ser, and His421Pro, respectively. Eight or more amino acid substitution mutations at eight or more positions functionally equivalent to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 of Bos taurus TdT of SEQ ID NO: 12 can include Glu191Val, Lys193Asn, Glu194Gly, Asp242Tyr, Lys287Glu, Phe296Leu, Met299Lys, Thr342Ser, and His421Pro, respectively.

[0067] In some embodiments, the recombinant TdT comprises eight amino acid substitution mutations at eight positions functionally equivalent to Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 of Bos taurus TdT of SEQ ID NO: 12. The eight amino acid substitution mutations at eight positions functionally equivalent to Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 of Bos taurus TdT of SEQ ID NO: 12 can be Lys193Asn, Glu194Gly, Asp242Tyr, Lys287Glu, Phe296Leu, Met299Lys, Thr342Ser, and His421Pro, respectively. In some embodiments, the recombinant TdT comprises nine amino acid substitution mutations at positions functionally equivalent to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 of Bos taurus TdT of SEQ ID NO: 12. The nine amino acid substitution mutations at positions functionally equivalent to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 of Bos taurus TdT of SEQ ID NO: 12 can be Glu191Val, Lys193Asn, Glu194Gly, Asp242Tyr, Lys287Glu, Phe296Leu, Met299Lys, Thr342Ser, and His421Pro, respectively.

[0068] thermal stability The recombinant TdT may be thermostable. The recombinant TdT may be stable at different temperatures in different embodiments. In some embodiments, the recombinant TdT may be stable at temperatures greater than or near 40° C., 41° C., 42° C., 43° C., 44° C., 45° C., 46° C., 47° C., 48° C., 49° C., 50° C., 51° C., 52° C., 53° C., 54° C., 55° C., 56° C., 57° C., 58° C., 59° C., 60° C., 61° C., 62° C., 63° C., 64° C., 65° C., 66° C., 67° C., 68° C., 69° C., 70° C., 71° C., 72° C., 73° C., 74° C., 75° C., 76° C., 77° C., 78° C., 79° C., 80° C., 81° C., 82° C., 83° C., 84° C., 85° C., 86° C., 87° C., 88° C., 89° C., or 90° C. For example, the recombinant TdT may be stable at temperatures greater than or equal to 47° C. The recombinant TdT may be stable at temperatures of 50° C. or higher. The recombinant TdT may be stable at temperatures of 55° C. or higher. The recombinant TdT may be stable at temperatures of 58° C. or higher. The recombinant TdT can be stable at a temperature of at least, at least about, at most, or at most about 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, or a number or range between any two of these values.

[0069] activation The terminal deoxynucleotidyl transferase activity of the recombinant TdT may be greater or less than that of bovine or Bos taurus TdT or fragments thereof. In some embodiments, the terminal deoxynucleotidyl transferase activity of the recombinant TdT is 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, or less than that of the Bos taurus TdT of SEQ ID NO: 12 or the recombinant TdT of SEQ ID NO: 14 at the same test temperature. %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124% ,125%,126%,127%,128%,129%,130%,131%,132%,133%,134%,135%,136%,137%,138%,139%,140%,141%,142%,143%,144%,145%,146%,147%,148%,149%,150%,151%,152%,153%,154%,155%,156%,157%,158%,159%,160%,161%,162%,163%,164%,165%,166%,167%,168%,169%,170%,171%,172%,173%,174%,175%,176%,177%,178%,179%,180%,181%,182%,183%,184%,185%,186%,187%,188%,189%,190%,191%,192%,193%,194%,195%,196%,197%,198%,199%,199%,190%,191%,192%,193%,194% 4%, 165%, 166%, 167%, 168%, 169%, 170%, 171%, 172%, 173%, 174%, 175%, 176%, 177%, 178%, 179%, 180%, 181%, 182%, 183%, 184%, 185%, 186%, 187%, 188%, 189%, 190%, 191%, 192%, 193%, 194%, 195%, 196%, 197%, 198%, 199%, 200% or more, or values ​​close to these.In some embodiments, the terminal deoxynucleotidyl transferase activity of the recombinant TdT is at least, at least about, at most, or at most about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154 %, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123 %, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%, 161%, 162%, 163%, 164%, 165%, 166%, 167%, 168%, 169%, 170%, 171%, 172%, 173%, 174%, 175%, 176%, 177%, 178%, 179%, 180%, 181%, 182%, 183%, 184%, 185%, 186%, 187%, 188%, 189%, 190%, 191%, 192%, 193%, 194%, 195%, 196%, 197%, 198%, 199%, 200%, or a number or range between any two of these values.For example, the terminal deoxynucleotidyl transferase activity of the recombinant TdT can be, or can be at least, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, or 120% of the terminal deoxynucleotidyl transferase activity of the Bos taurus TdT of SEQ ID NO: 12 or the terminal deoxynucleotidyl transferase activity of the recombinant TdT of SEQ ID NO: 14 at the same test temperature.

[0070] The test temperature may vary in different embodiments. In some embodiments, the test temperature is 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, 99°C, 100°C, 101°C, The test temperature may be greater than or near 37°C, 47°C, 50°C, 55°C, or 58°C. In some embodiments, the test temperature is at least, at least about, at most, or at most about 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, The temperature may be 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, or a number or range between any two of these values.

[0071] Additional Components In some embodiments, the recombinant TdT comprises a purification tag, such as a His-tag or glutathione S-transferase. The purification tag can be at the N-terminus of the recombinant TdT, the C-terminus of the recombinant TdT, or internal to the recombinant TdT. The recombinant TdT can comprise a protease cleavage sequence, such as LeuValProArg / GlySer (thrombin cleavage site) or LeuGluValLeuPheGln / GlyPro (PreScission protease cleavage site), between the purification tag and another component (e.g., a Bos taurus TdT fragment) or the remainder of the recombinant TdT.

[0072] In some embodiments, the recombinant TdT comprises a small ubiquitin-like modifier (SUMO) protein, or a fragment thereof. The sequence of the SUMO protein or fragment thereof in the recombinant TdT can vary in different embodiments. In some embodiments, the SUMO protein or fragment thereof in the recombinant TdT is a SUMO protein (e.g., suppressor of mif two 3 in yeast, SMT 3), or a fragment thereof (e.g., a SUMO fragment comprising the amino acid sequence of SEQ ID NO: 13) that is 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or thereabouts identical thereto. For example, the SUMO fragment in the recombinant TdT may comprise an amino acid sequence at least 80% identical to SEQ ID NO: 13. In some embodiments, the SUMO protein or fragment thereof in the recombinant TdT may be a SUMO protein (e.g., suppressor of mif two 3 in yeast, SMT 13), or a fragment thereof (e.g., a SUMO fragment comprising the amino acid sequence of SEQ ID NO: 13).

[0073] The position of the SUMO fragment in the recombinant TdT may vary in different embodiments. In some embodiments, the recombinant TdT comprises a SUMO fragment at the N-terminus of the recombinant TdT. In some embodiments, the recombinant TdT comprises a SUMO fragment at the C-terminus of the recombinant TdT.

[0074] The recombinant TdT can comprise an amino acid sequence having sequence identity or about a threshold sequence identity to a recombinant TdT comprising a SUMO fragment (e.g., a recombinant TdT having a SUMO fragment comprising the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15). The recombinant TdT can comprise an amino acid sequence having sequence identity above, about, below, or about below a threshold sequence identity to a recombinant TdT comprising a SUMO fragment (e.g., SEQ ID NO: 14 or SEQ ID NO: 15). The recombinant TdT can comprise an amino acid sequence having at least, at least about, at most, or at most about the threshold sequence identity to a recombinant TdT comprising a SUMO fragment (e.g., SEQ ID NO: 14 or SEQ ID NO: 15). The threshold sequence identity can be different in different embodiments. In some embodiments, the sequence identity threshold is 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or range between any two of these values. For example, the recombinant TdT can comprise an amino acid sequence that is at least 80% identical to SEQ ID NO: 14. As another example, the recombinant TdT can comprise an amino acid sequence that is at least 80% identical to SEQ ID NO: 15.

[0075] Mutant terminal deoxynucleotidyl transferase In the present disclosure, various types of mutagenesis can be used to modify TdT to create variants, for example, according to TdT models and model predictions, or using random or semi-random mutation approaches. Generally, any available mutagenesis procedure can be used to create TdT variants. Such mutagenesis procedures can include selection of mutant nucleic acids and polypeptides for one or more desired activities (e.g., enhanced seeding and / or amplification on a solid support). Procedures that can be used include, but are not limited to, site-directed point mutagenesis, random point mutagenesis, in vitro or in vivo homologous recombination (DNA shuffling and combinatorial overlap PCR), mutagenesis using uracil-containing templates, oligonucleotide-directed mutagenesis, phosphorothioate-modified DNA mutagenesis, mutagenesis using gapped duplex DNA, point mismatch repair, mutagenesis using repair-deficient host strains, restriction-selection and restriction-purification, deletion mutagenesis, mutagenesis by total gene synthesis, degenerate PCR, double-strand break repair, and many others known to those skilled in the art. The starting TdT for mutation can be any of those described herein.

[0076] Mutagenesis can be guided by known information from naturally occurring TdT molecules or known information of known altered or mutated TdTs, such as sequences, sequence comparisons, physical properties, crystal structures, and / or others described herein. However, in another class of embodiments, the modifications can be essentially random (e.g., as in classical or "family" DNA shuffling).

[0077] Production and isolation of recombinant terminal deoxynucleotidyl transferase Generally, polynucleotides or nucleic acids encoding the recombinant TdT provided herein can be produced by cloning, recombination, in vitro synthesis, in vitro amplification, and / or other available methods. A variety of recombinant methods can be used to express the expression vectors encoding the recombinant TdT provided herein. Methods for producing recombinant nucleic acids, and expression and isolation of expression products are known. Numerous exemplary mutations and combinations of mutations, as well as strategies for designing desirable mutations, are described herein.

[0078] Additionally, kits for purifying plasmids or other related nucleic acids from cells are commercially available. Any isolated and / or purified nucleic acid can be further manipulated to produce other nucleic acids used to transfect cells, incorporated into related vectors, transfect organisms for expression, and / or otherwise. Typical cloning vectors contain transcription and translation terminators, transcription and translation initiation sequences, and promoters useful for regulating expression of a specific target nucleic acid. The vector optionally contains a generic expression cassette containing at least one independent terminator sequence, sequences allowing replication of the cassette in eukaryotes, prokaryotes, or both (e.g., shuttle vectors), and selectable markers in both prokaryotic and eukaryotic systems. The vector is suitable for replication and integration in prokaryotes, eukaryotes, or both.

[0079] A particular amino acid can be encoded by multiple codons, and particular translation systems (e.g., prokaryotes or eukaryotes) often exhibit codon bias; for example, different organisms often prefer one of several synonymous codons that encode the same amino acid. Thus, the polynucleotides or nucleic acids presented herein can be "codon-optimized," meaning that the polynucleotides or nucleic acids are synthesized to contain codons preferred by the particular translation system used to express recombinant TdT. For example, if it is desired to express recombinant TdT in a bacterial cell (or even a particular strain of bacteria), the polynucleotides or nucleic acids can be synthesized to contain the codons most frequently found in the genome of that bacterial cell for efficient expression of recombinant TdT. A similar strategy can be used if it is desired to express recombinant TdT in a eukaryotic cell, e.g., the nucleic acid contains codons preferred by that eukaryotic cell.

[0080] A variety of protein isolation and detection methods are known and can be used, for example, to isolate recombinant TdT from recombinant cultures of cells expressing the recombinant TdT presented herein. A variety of protein isolation and detection methods are known. Recombinant TdT can be isolated and detected as disclosed herein.

[0081] Nucleic acid encoding recombinant terminal deoxynucleotidyl transferase Disclosed herein are embodiments of polynucleotides encoding any of the recombinant terminal deoxynucleotidyl transferases (TdTs) of the present disclosure. Disclosed herein are embodiments of expression vectors comprising a polynucleotide encoding any of the recombinant terminal deoxynucleotidyl transferases (TdTs) of the present disclosure.

[0082] Further provided herein are nucleic acid molecules (e.g., polynucleotides) encoding the TdTs provided herein. For any given modified TdT that is a variant of TdT from a species whose amino acid sequence, preferably a wild-type nucleotide sequence encoding TdT, is also known, a nucleotide sequence encoding the variant can be obtained according to basic principles of molecular biology. For example, assuming that the wild-type nucleotide sequence encoding TdT is known, it is possible to use the standard genetic code to deduce the nucleotide sequence encoding any given variant of TdT with one or more amino acid substitutions. Similarly, nucleotide sequences can be easily derived for variants of TdT from other species. A nucleic acid molecule having the required nucleotide sequence can then be constructed using standard molecular biology techniques.

[0083] According to the embodiments provided herein, the defined nucleic acids include not only identical nucleic acids but also any minor base variations, including substitutions resulting in synonymous codons (different codons specifying the same amino acid residue) due to degenerate coding, particularly conservative amino acid substitutions. The term "nucleic acid sequence" also includes complementary sequences to any single-stranded sequence given in terms of base variations.

[0084] The nucleic acid molecules described herein may also advantageously be included in a suitable expression vector for expressing the TdT encoded therefrom in a suitable host. The incorporation of cloned DNA into a suitable expression vector for subsequent transformation of the cells and subsequent selection of transformed cells is well known.

[0085] Such expression vectors include vectors having a nucleic acid according to embodiments presented herein operably linked to a regulatory sequence, such as a promoter region, capable of effecting expression of the DNA fragment. The term "operably linked" refers to a juxtaposition wherein the described components are in a relationship permitting them to function in their intended manner. Such vectors can be transformed into a suitable host cell to provide for expression of a protein according to embodiments presented herein.

[0086] The nucleic acid molecule may encode a mature protein or a protein having a prosequence (including one encoding a leader sequence on the preprotein), which is then cleaved by the host cell to form the mature protein. The vector may be, for example, a plasmid, virus, or phage vector provided with an origin of replication, optionally a promoter for expression of the nucleotide, and optionally a regulator of the promoter. The vector may contain one or more selectable markers, such as, for example, an antibiotic resistance gene.

[0087] Regulatory elements required for expression include a promoter sequence for binding RNA polymerase and directing an appropriate level of transcription initiation and a translation initiation sequence for ribosome binding. For example, a bacterial expression vector may contain a promoter such as the lac promoter, as well as a Shine-Dalgarno sequence and the start codon AUG for translation initiation. Similarly, a eukaryotic expression vector may contain a heterologous or homologous promoter for RNA polymerase II, a downstream polyadenylation signal, the start codon AUG, and a termination codon for ribosome detachment. Such vectors may be commercially available or may be assembled from the described sequences by methods well known in the art.

[0088] Transcription of DNA encoding TdT by higher eukaryotes can be optimized by including an enhancer sequence in the vector. Enhancers are cis-acting elements of DNA that act on promoters to increase transcription levels. Vectors also generally contain an origin of replication as well as a selectable marker.

[0089] cell Disclosed herein are embodiments of host cells comprising any of the recombinant terminal deoxynucleotidyl transferases of the present disclosure. Disclosed herein are embodiments of host cells comprising a polynucleotide encoding any of the recombinant terminal deoxynucleotidyl transferases (TdT) of the present disclosure. Disclosed herein are embodiments of host cells comprising an expression vector comprising a polynucleotide encoding any of the recombinant terminal deoxynucleotidyl transferases of the present disclosure.

[0090] kit Disclosed herein include kits. In some embodiments, the kits comprise a recombinant terminal deoxynucleotidyl transferase (TdT) of the disclosure and instructions for using the recombinant terminal deoxynucleotidyl transferase. In some embodiments, the kits comprise a polynucleotide encoding a recombinant terminal deoxynucleotidyl transferase of the disclosure and instructions for using the polynucleotide and / or recombinant terminal deoxynucleotidyl transferase. In some embodiments, the kits comprise an expression vector comprising a polynucleotide encoding a recombinant terminal deoxynucleotidyl transferase of the disclosure and instructions for using the expression vector, polynucleotide, and / or recombinant terminal deoxynucleotidyl transferase. In some embodiments, the kit comprises a host cell comprising a recombinant terminal deoxynucleotidyl transferase of the present disclosure, a polynucleotide encoding the recombinant terminal deoxynucleotidyl transferase, an expression vector comprising a polynucleotide encoding the recombinant terminal deoxynucleotidyl transferase, or a combination thereof, and instructions for using the recombinant terminal deoxynucleotidyl transferase, the polynucleotide, the expression vector, the host cell, or a combination thereof.

[0091] Example Certain aspects of the above-described embodiments are disclosed in further detail in the following examples, which are not intended to limit the scope of the disclosure.

[0092] Example 1 Evolution of thermostable terminal deoxynucleotidyl transferases Terminal deoxynucleotidyl transferase (TdT) catalyzes the template-free incorporation of any nucleotide into single-stranded DNA. This unique feature has led to its widespread use in biotechnology and clinical applications. One possible use is the synthesis of long de novo DNA molecules by TdT-mediated iterative incorporation of 3' reversibly blocked nucleotides followed by subsequent deblocking. However, wild-type (WT) TdT is not optimized for the incorporation of 3'-modified nucleotides, and TdT engineering is hindered by the fact that TdT is only marginally stable and present only in mesophilic organisms. This example describes the evolution of a thermostable TdT variant to serve as a scaffold for subsequent evolution to enable efficient incorporation of 3'-modified nucleotides. Because evolution to incorporate bulky modified nucleotides generally results in decreased TdT stability, a thermostable variant would be a good starting point for such efforts. Furthermore, thermostable TdT would be useful even in cases where DNA secondary structures inhibit WT activity, as higher temperatures can be used to melt dsDNA. We developed an assay to identify thermostable TdT variants. After screening approximately 10,000 TdT mutants, we found a variant, designated TdT3-2, that was 10°C more thermostable than SUMO-TdT while retaining the catalytic properties of TdT.

[0093] Materials and Methods Establishment of a plate-based FRET assay Oligonucleotide 2bA FAM (5'-CGC TTG CAC AGG TGC GTT / iFluorT / CA-3', SEQ ID NO: 16) consisted of fluorescein (FAM) dye on the T base and two bases from the 3'-OH were purchased from Integrated DNA Technologies (IDT, Coralville, Iowa, USA). Cy5-dCTP (NU-809-CY5) was purchased from Jena Bioscience (Germany). Terminal transferase (NEB TdT) was purchased from New England Biolabs (NEB, Ipswich, Massachusetts, USA).

[0094] Signal acquisition using FAM and Cy5 Förster or fluorescence resonance energy transfer (FRET) pairs was tested using 200 nM 2bA FAM oligo, 600 nM Cy5-dCTP, and 20 U NEB TdT in 1X TdT buffer (200 mM potassium acetate, 25 mM Tris, 0.01% (v / v) Triton X-100, 1 mM cobalt chloride, pH 7.2) in a final volume of 50 μL. A negative control without NEB TdT was included. For incorporation of Cy5-dCTP into the 2bA FAM oligo by TdT, reaction samples were incubated at room temperature for 1 min. Reactions were quenched by heating at 80°C for 5 min. Samples were transferred to black-bottom 384-well plates (Greiner, Austria) for signal detection on a Biotek Synergy H1 Hybrid Multi-mode reader (Winooski, VT, USA). The excitation was set at 450 nm, and the emission wavelength was scanned from 480 nm to 700 nm.

[0095] In this disclosure, SUMO-TdT refers to recombinant TdT containing amino acids 139-520 of bovine (Bos taurus) TdT and an N-terminal SUMO tag to improve solubility and expression. Table 1 shows the sequence of SUMO-TdT. Figure 1 shows a non-limiting exemplary sequence alignment of SUMO-TdT with amino acids 139-520 of Bos taurus TdT. The pET28b plasmid carrying SUMO-TdT was transformed into Escherichia coli (E. coli) BL21(DE3) (NEB, USA) according to the manufacturer's protocol and induced with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) (Sigma, St. Louis, MO, USA) overnight at 15°C in a 250 rpm shaker. Lysates of induced TdT-expressing cells were divided into four portions (undiluted, 10-fold diluted, 100-fold diluted, and 1000-fold diluted with 1X TdT buffer) and lysed using a Q700 horn sonicator (Qsonica, Newtown, CT, USA). Oligo FAM20 (5'- / 56-FAM / CGCTTGCACAGGTGCGTTCG-3', SEQ ID NO: 17) (IDT, USA) and deoxycytidine triphosphate (dCTP (Invitrogen, Waltham, MA, USA)) were added to the lysed and diluted cell lysates to achieve final cell lysate dilutions of 5x, 50x, 500x, and 5000x. For TdT-mediated incorporation of dCTP into FAM20, the samples were incubated at 37°C for 1 min. The reaction solution was heated at 80° C. for 5 minutes, and the reaction was quenched by adding an equal volume of 2× TBE-urea sample buffer (Invitrogen, USA).

[0096] Quenched reaction samples were analyzed on a 20% polyacrylamide gel. S2 glass plates (Apogee, Baltimore, MD, USA) were washed with Alconox and water, then wiped with ethanol, water, 5% dichlorodimethylsilane (DCDMS) (Sigma, USA), and water. The plates were assembled with 0.8 mm spacers (Apogee, USA), gel seal tape (Apogee, USA), and binder clips. A 1 mL solution of 0.2% (v / v) ammonium persulfate (APS) (Sigma, USA) and 0.2% (v / v) tetramethylethylenediamine (TEMED) (Bio-Rad, Hercules, CA, USA) in water was prepared and poured onto the assembled glass plates. A 20% acrylamide solution (90 mL) was prepared by mixing 45 mL of 40% acrylamide / bis 19:1 solution (Bio-Rad, USA) and 45 mL of water, followed by the addition of 225 μL of 10% APS and 22.5 μL of TEMED. The 20% acrylamide solution was loaded into a glass plate using a 50 mL syringe. A 0.8 mm-thick 32-well plastic comb (Apogee, USA) was inserted into the gel and allowed to polymerize for 1 hour and 30 minutes. After carefully removing the comb and gel tape, the gel plate was attached to an S2 Sequencing Gel Electrophoresis Apparatus (Biometra-Analytik Jena, Germany). 1X Tris / borate (TBE) running buffer was poured into the top and bottom reservoirs to a depth of approximately 3 cm above the gel. Gel fragments in the wells were removed. Before loading the samples, pre-electrophoresis was performed at 1700 V for 15 min to obtain a gel surface temperature of approximately 50°C. 15 μL of each quench reaction sample was loaded into each well and run for 1 h 30 min at 1700 V. Gels were imaged on a Bio-Rad Gel Doc XR+ (USA) using Sybr Green exposure.

[0097] As shown in Figure 6, we established a workflow for a plate-based FRET assay. To examine the robustness of this assay, a blind study was performed. An empty pET28b vector and a SUMO-TdT-expressing Escherichia coli (E. coli) BL21(DE3) (New England Biolabs, USA) colony were inoculated into 200 μL of Luria Broth (LB) supplemented with 50 μg / mL kanamycin in each well of a 96-deep-well plate (Eppendorf, Germany) and grown overnight at 37°C and 250 rpm. The grown culture was subcultured 100-fold into 495 μL of fresh LB supplemented with 50 μg / mL kanamycin in a new 96-deep-well plate. The culture was incubated at 24°C for 4 hours and then induced with 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) at 24°C and 800 rpm for at least 16 hours. The induced cell lysate was diluted 10-fold with 1X TdT buffer in a 0.2 mL PCR tube plate (AITbiotech, Singapore). The cell lysate was lysed using a horn sonicator with the following parameters: Amp 90, 3 min process time, 5 s pulse on, and 15 s pulse off. 24 μL of a master mix containing 200 nM 2bA FAM oligo, 600 nM Cy5-dCTP, and 1X TdT buffer was added to each well in a black 384-well plate. A suspension of lysed cells (6 μL) was added to each well. FRET signal detection was performed for 30 min on an EnSpire Multimode plate reader (Waltham, MA, USA) with excitation at 450 nm and emission detection at 670 nm.

[0098] Generation of TdT mutant library The SUMO-TdT plasmid was used as a backbone template for mutagenesis. Using the error-prone polymerase mutazyme II (Agilent Technologies, USA), gene primer 1 (5'-AAAAAACACCTGCGGCCTGGTGCCGCGCGGCAGCGCTAGCATG-3', SEQ ID NO: 18) and gene primer 2 (5'-AAAAAACACCTGCGGGATCCGGTACCGCGGCCGCCTA-3', SEQ ID NO: 19) were used to generate a library of 2-5 mutations per gene length on average. The mutagenized pool was ligated into the pET28b vector and transformed into Escherichia coli (E. coli) 10-β competent cells (New England Biolabs, USA) for plasmid amplification. Cells were harvested and scaled up in 50 mL of LB supplemented with 50 μg / mL kanamycin to reach an OD of 0.01. 600nm The cells were grown at 37°C until a RI of ~0.5 was achieved. The cell culture was harvested and the plasmid was extracted using a Miniprep kit (Qiagen, Germany). The same mutagenesis process was performed using TdT1-1 as the parental template for the second round of screening.

[0099] The mutant library for the third round of screening used TdT1-1 and TdT1-3 as parental templates to incorporate different combinations of mutations identified from TdT2-1, TdT2-2, TdT2-3, and TdT2-4. Primers used to introduce mutations into the parental templates: Primer A: 5'-AATTCTGTGTTTAAWGRAAATGAAGTCTCTTATGTG-3' (SEQ ID NO: 20) Primer B: 5'-AGATCTCTGAGTRAAATAATGTCAGACAAAACCCTGAA-3' (SEQ ID NO: 21) Primer C: 5'-AGACAAAACCCTGAAATTMACAAAAAWGCAGAAAGCAGGAT-3' (SEQ ID NO: 22) Primer D: 5'-TTTGTCACCATGWCAGGAGGATTCCGCAG-3' (SEQ ID NO: 23) Primer E: 5'-GATTTTAAAATTGCMCCATCAGAGAGTAGACAGT-3' (SEQ ID NO: 24)

[0100] Screening for potentially thermostable TdT mutants The amplified mutant library plasmids were transformed into the expression strain Escherichia coli (E. coli) BL21(DE3). The recovered cells were plated on LB agar supplemented with 50 μg / mL kanamycin and incubated overnight at 37 °C. Individual colonies were picked and inoculated into 200 μL of LB supplemented with 50 μg / mL kanamycin in a 96-deep-well plate. Growth cell culture and downstream processes were performed as described above under "Establishment of a plate-based FRET assay" (Figure 6). Diluted and lysed cell lysates were subjected to different heat treatments and then added to 2bA FAM oligo and Cy5-dCTP.

[0101] For the first round of screening using the SUMO-TdT template-based mutant library, the diluted and lysed cell lysate was divided into two samples: one was not heat-treated and the other was subjected to 47°C for 1 min. A library size of 2790 mutants was screened.

[0102] In the second round of thermostability screening, TdT1-1 identified from round 1 was used as a template for mutagenesis. A library size of 7356 was screened with an aliquot of each cell lysate that had not been subjected to heat treatment, and the remaining aliquot was heat-treated at 50°C for 1 min.

[0103] One of the mutant libraries for the third round of thermostability screening was created using degenerate primers consisting of either the WT sequence or mutations identified from the top four mutants from round 2 (TdT2-1, TdT2-2, TdT2-3, and TdT2-4) based on the TdT1-1 template, resulting in a combinatorial mixed pool. A library size of 736 was screened using an aliquot of each cell lysate sample without heat treatment and the remaining aliquot subjected to 55°C for 1 minute. The same was done for the TdT1-3 template-based mutant library using an aliquot of each cell lysate sample without heat treatment and the remaining aliquot subjected to 58°C for 1 minute.

[0104] The top mutant(s) from each library were identified. The genes were prepared for sequencing. Information about the resulting mutations was inserted into the parental plasmid and retransformed into the expression E. coli strain. Approximately five or six colonies of the same mutant were selected and subjected to replicate FRET assays. Average data from the average FRET signal read over 15 to 30 minutes for each mutant were obtained and plotted. The activity ratio for each TdT was obtained by dividing the average FRET readout under heat treatment by the average FRET readout without heat treatment. Student's T test assuming unequal variances determined significant differences between the average FRET signals from the parental and mutant TdT.

[0105] Purification of TdT Plasmids containing SUMO-TdT and TdT3-2 were expressed in E. coli BL21(DE3). Each E. coli construct was grown in 6 L 2xYT broth (Bio Basic, Canada) supplemented with 50 μg / mL kanamycin. OD 600nmAt ~0.8, the cell culture was induced with 1 mM IPTG. Induction was carried out at 250 rpm for at least 16 hours at 15°C. The induced cells were harvested by centrifugation at 6,500 rpm for 6 minutes at 4°C in a 500 mL Beckman Coulter plastic vessel using a Beckman Coulter (Brea, CA, USA) JLA10.500 rotor. The cell pellet was resuspended in a total of 120 mL of binding buffer (20 mM Tris-HCl (pH 7.9), 500 mM NaCl, and 5 mM imidazole) at 4°C. The resuspended cells were lysed by five passes at 20,000 psi using a microfluidizer (Microfluidics International Corporation, Westwood, MA, USA). After centrifugation at 20,000 rpm for 20 minutes at 4°C using a Beckman Coulter JA25.5 rotor, the supernatant was collected. Ni was measured on an AKTA Pure instrument (GE Healthcare Life Science, Chicago, IL, USA). 2+ His-tagged TdT was purified from the supernatant using a Chelating Sepharose Fast Flow column (Pharmacia Biotech, Piscataway, NJ, USA) packed with 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Samples from several fractions were analyzed on a 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel. Fractions containing the desired band corresponding to TdT were pooled and dialyzed against Buffer 1 (20 mM Tris-HCl, pH 8, 100 mM NaCl, 100 mM histidine, 0.1 mM 2,2'-bipyridyl) using a 10 kD molecular weight cutoff (MWCO) dialysis membrane (Spectrum Chemical, USA) at 4°C for at least 6 h. Buffer 1 was then exchanged for Buffer 2 (50 mM Tris-HCl, pH 8) for at least 4 h at 4°C, after which the dialyzed protein was loaded onto a 10 mL Q Sepharose column (GE Healthcare Life Sciences). Elution was performed over 1 CV (1 M NaCl). 280nmSmall aliquots of the fractions containing the expected bands were analyzed on a 15% SDS-PAGE gel. Fractions containing the expected bands corresponding to each TdT were pooled and dialyzed against protein storage buffer (20 mM Tris-HCl, pH 8, 100 mM NaCl). The dialyzed protein was aliquoted, frozen in liquid nitrogen, and stored at -80°C.

[0106] The purity of purified SUMO-TdT and TdT3-2 was analyzed via a nuclease assay. Each protein (0.5 μg) was incubated with 1 μg of single-stranded DNA (5′-TCTAGAGCCCGCCTAATCAGCGGGCTTTTTTTTAT-3′, SEQ ID NO: 25) in 1× Tris / acetate (TA) buffer (30 mM Tris, 66 mM potassium acetate, 10 mM magnesium acetate, 0.5 mM DTT, pH 7.8) at 37°C for 16 hours. A negative control using a nuclease-free enzyme, such as a polymerase sample from Illumina, Inc. (San Diego, CA, USA), and a positive control using DNase I (New England Biolabs, USA) were included. Samples were visualized on a 15% TBE-urea gel (Invitrogen, USA).

[0107] Determination of thermal stability of TdT3-2 Differential scanning calorimetry (DSC) (TA Instruments, New Castle, DE, USA) was performed to examine the thermal stability of SUMO-TdT and TdT3-2. A temperature range of 20°C to 80°C and a scan rate of 60°C / h were used. SUMO-TdT and TdT3-2 were diluted to approximately 1 mg / mL with 1X TdT buffer, and 1 mL of each protein was loaded onto the DSC. Duplicates of each protein were loaded, and the average heat capacity was plotted.

[0108] Nano differential scanning fluorimetry (DSF) (NanoTemper, Germany) was performed to verify the thermal stability of SUMO-TdT and TdT3-2. The excitation power was set to 14% with a scan rate of 60 °C / h from 20 °C to 80 °C. Fluorescence measurements were performed at an excitation wavelength of 470 nm. The DSF signal ratio F350 / F330 was calculated from the emission intensities at 330 nm (F330) and 350 nm (F350). Light scattering measurements were performed in parallel. Approximately 1 mg / mL of each protein sample (10 μL) was loaded into the nanoDSF, and duplicates of each sample were measured. The average of the first derivative of the ratio F350 / F330 was plotted.

[0109] To verify the thermal stability of SUMO-TdT and TdT3-2, a SYPRO Orange thermal shift assay was performed. Duplicate 4 μg of each protein containing a final 5X SYPRO Orange dye (Life Technologies, Carlsbad, CA, USA) was used in the assay. A temperature range of 4°C to 95°C with an increment of 0.5°C per 30 seconds was programmed. The change in relative fluorescence (RFU) versus temperature (-dRFU / temperature) was obtained, and the average was plotted against temperature.

[0110] Circular dichroism (CD) was used to evaluate the thermal stability of SUMO-TdT and TdT3-2. A Chirascan (Applied Photophysics, United Kingdom) Q100 with a 0.1 mm manual quartz cuvette was used for this study. Protein unfolding was monitored as a function of temperature at wavelengths from 198 nm to 260 nm, and a wavelength of 222 nm was used for data analysis. Samples were diluted with 1X TdT buffer to obtain a final protein concentration of 1.6 mg / ml. The temperature gradient was set from 25°C to 70°C at a rate of 1°C / min with a tolerance of 0.2°C. The instrument bandwidth was set to 1 nm with an acquisition time of 1 second per point. Two separate measurements were performed for each condition. The average of the first derivative of CD (222 nm) was plotted.

[0111] Endpoint assays were performed to verify the activity of SUMO-TdT and TdT3-2 at 25°C, 36°C, 47°C, and 58°C. NEB TdT (5 units) and purified SUMO-TdT and TdT3-2 (100 nM each) were incubated with 200 nM FAM oligo (5'- / 56-FAM / ATTCAGGACGAGCCTCAGACC-3', SEQ ID NO: 26), 5 μM dCTP, and 1X TdT buffer at 25°C, 36°C, 47°C, and 58°C for 5, 10, and 20 minutes, respectively. The reaction was terminated by subjecting the samples to 90°C for 5 minutes. An equal volume of 2X TBE-urea sample buffer was added to each sample before heating to 80°C for 5 minutes. Samples were visualized on a 15% TBE-urea gel (240V, 62 minutes). The gel was imaged with a Gel Doc XR+ (Bio-Rad, USA).

[0112] The activity of commercially available NEB TdT and TdT3-2 on blunt-ended DNA substrates was tested. The blunt-ended DNA substrate consisted of a primer (5'- / 56-FAM / TTTCGGTGGTCGCCGTATCCGC-3', SEQ ID NO: 27) and a template (5'-GCGGATACGGCGACCACCGAGATCTACACTCTGAG / 3Phos / -3', SEQ ID NO: 28) (IDT, USA). Double-stranded DNA (ds DNA) products were prepared by heating a mixture of 4 μM DNA (primer to template, 1:3) and 1X TdT buffer at 95°C for 5 minutes and then cooling to room temperature for at least 15 minutes. The enzyme (10 μL) and reaction mixture (30 μL) were equilibrated to their respective reaction temperatures for 30 seconds. The enzyme was then added to a reaction mixture containing 200 nM dsDNA, 50 μM 2',3'-dideoxycytidine 5'-triphosphate (ddCTP) (Jena Bioscience, Germany), 1X TdT buffer, and 0.5 μM enzyme (or 25 U / 20 μL reaction for NEB TdT), and the mixture was heated at 37°C to 50°C for 1, 2, and 4 min in equal portions. Samples (10 μL) from each time point were quenched by mixing with 8.3 μM displacer oligo and 2X TBE-urea sample dye (Invitrogen, USA) (12 μL), heating at 95°C for 5 min, and then cooling to room temperature. A displacer oligo (5'-TTTCTCAGAGTGTAGATCTCGGTGGTCGCCGTATCCGC / 3Phos / -3', SEQ ID NO: 29) (IDT, USA) anneals to the template, allowing the primer to be displaced and present as ssDNA, while the sample buffer and high temperature denature the enzyme. Samples were visualized on a 15% TBE-urea gel (240 V, 68 min), and the gel was imaged with GelDoc. Quantification of bands in each sample was completed using Image Lab software (Bio-Rad, USA). Reactions were performed in duplicate, and the average percentage of primers with ddCTP incorporation was plotted using GraphPad (GraphPad Software, Inc., San Diego, CA, USA).

[0113] Kinetic studies of SUMO-TdT and TdT3-2 Reaction assays were set up in a final volume of 20 μL using 200 nM FAM oligo, various dCTP concentrations, and 86 nM SUMO-TdT and TdT3-2. Incorporation of dCTP by TdT was carried out at 37°C for 15 s. The reaction was quenched by adding 10 mM EDTA and an equal total sample volume of 2X TBE-urea sample buffer and heating to 80°C for 5 min. Samples were loaded onto a 15% TBE-urea gel and run at 180 V for 1 h 30 min. Gels were imaged using a Gel Doc XR+, and bands were analyzed using Image Lab. The band percentage of each band within a lane was obtained from the software and converted to the amount of FAM oligo corresponding to each band. Band size provided information about the number of dCTP incorporations carried out by TdT, allowing calculation of the total metabolic turnover in nM for a 20 μL reaction. The resulting values ​​were converted to μM to obtain enzyme turnover rates in μM dCTP / s. Enzyme turnover rates were plotted against the concentration of dCTP in GraphPad Prism 8 using Michaelis-Menten analysis.

[0114] The kinetics of SUMO-TdT and TdT3-2 were also tested at 47°C. Reactions were set up with 200 nM FAM oligo, 173 nM SUMO-TdT and TdT3-2, and various dCTP concentrations (final 20 μL). dCTP incorporation was carried out for 5 s at 47°C. Subsequent quenching and analysis were identical to the kinetics performed at 37°C in the previous paragraph.

[0115] Modeling the 3D structure of TdT3-2 The amino acid sequence of TdT3-2 (Table 1) was entered into the Phyre2 web portal (http: / / www.sbg.bio.ic.ac.uk / phyre2 / html / page.cgi?id=index). The "NORMAL" mode was selected. The 3D structure was modeled.

[0116] [Table 1]

[0117] Results and Discussion Establishment of a plate-based FRET assay for the detection of TdT activity The high-throughput assay for screening thermostable TdT variants described in this example is a Förster resonance energy transfer (FRET)-based assay that determines TdT activity in covalently linking primers to nucleotides (Figures 2 and 3A). First, the optimal distance between the donor and acceptor dyes must be determined. We utilized the FAM-TAMRA FRET pair, varying the distance of the FAM from the 3' end. Positioning the FAM donor dye two bases away from the 3' end of the oligo resulted in the greatest decrease in FAM emission. However, the corresponding increase in emission from carboxytetramethylrhodamine (TAMRA) was low (Figure 4, Table 2). To obtain a higher acceptor emission signal, different FRET pairs (FAM-Cy5 and Cy3-Cy5) were tested while maintaining the optimal two-base distance from the 3' end (Figure 4, Table 3). FAM-Cy5 resulted in the greatest increase in signal detected from the acceptor Cy5 emission at 670 nm (Figure 3B and Figures 5A-5C). A primer with FAM two bases from the 3' end (2bA FAM) and Cy5-dCTP was used for subsequent screening of active TdT mutants (Fig. 3A).

[0118] [Table 2]

[0119] [Table 3]

[0120] For practical high-throughput screening, assays would be best performed using crude lysates because the purification step adds time and cost burdens. However, utilizing crude cell lysates was problematic because the presence of endogenous Escherichia coli (E. coli) nucleases could mask TdT activity (Figure 3C, top row). One potential way to overcome this problem was to dilute the cell lysates. Without being bound by any particular theory, for example, if TdT has a higher binding affinity for the oligosubstrate, or if overexpression of TdT results in a higher concentration of TdT compared to the nuclease after dilution of the cell lysate, diluting the cell lysate could disproportionately reduce nuclease activity relative to TdT (Figure 3C, bottom row). To determine the validity of this hypothesis, cell lysates expressing SUMO-TdT were diluted 5, 50, 500, and 5,000-fold, and then the integration reactions were analyzed on a polyacrylamide gel. Although bands corresponding to +1 or higher were observed for 50- and 500-fold diluted cell lysates, nuclease activity predominated at a 5-fold dilution (Figure 3D). Along with the FAM-Cy5 FRET pair described above, a 50-fold dilution factor was chosen for thermostability screening.

[0121] To determine the robustness of the resulting plate-based FRET assay, colonies expressing either SUMO-TdT or the empty plasmid (48 colonies each) were inoculated into a 96-deep-well plate, and the respective lysates were assayed after 50-fold dilution using the previously developed FRET assay. The SUMO-TdT-expressing cell lysate had a higher FRET readout and could be distinguished from the negative control (Figure 3E). Based on this result, a workflow for a 96-well plate-based FRET assay to screen potentially thermostable TdT mutants was established, as shown in Figure 6. This includes an initial heat shock step to distinguish between thermostable and thermosensitive mutants. Note that the selection pressure can be adjusted by changing the temperature and duration of the heat shock step.

[0122] Discovery of heat-stable TdT3-2 Mutant libraries were generated via random mutagenesis. The first mutant library used SUMO-TdT (i.e., bovine TdT lacking the N-terminal 138 amino acids and containing an N-terminal SUMO tag) as the parent template. The 2790 mutant library was screened by heat treatment at 47°C for 1 minute. This round identified the thermostable mutants TdT1-1 and TdT1-2 as thermostable (Figure 2 and Tables 4A and 4B). TdT1-1 and TdT1-2 have significantly higher FRET readouts without and with heat treatment compared to SUMO-TdT (Figure 7A). Furthermore, TdT1-1 and TdT1-2 retained a high percentage of their activity after heat treatment (Figure 7A).

[0123] [Table 4]

[0124] [Table 5]

[0125] In the second round of screening, TdT1-1 was used as the parent to generate a library size of 7636. TdT1-1 was chosen as the parent template because it had a higher FRET readout without heat treatment compared to TdT1-2. A second round of screening was performed with heat treatment at 50°C for 1 minute, identifying four thermostable mutants (TdT2-1, TdT2-2, TdT2-3, and TdT2-4) (Tables 4A and 4B). The four mutants have significantly higher FRET readouts than TdT1-1 under both the non-heat-treated and heat-treated conditions (Figure 7B). All four mutants also retained a much higher percentage of their activity after 1 minute at 50°C (Figure 7B).

[0126] It was predicted that the combination of mutations identified from TdT2-1, TdT2-2, TdT2-3, and TdT2-4 would result in a synergistic increase in thermostability. In the third round of thermostability screening, two mutant libraries were generated. One of the mutant libraries was based on the TdT1-1 template, which contained different combinations of mutations found in TdT2-1, TdT2-2, TdT2-3, and TdT2-4. The other mutant libraries were generated similarly, except that TdT1-3 was used as the parent template. TdT1-3 consisted of a combination of mutations in TdT1-1 and TdT1-2 (Tables 4A and 4B). The TdT1-1-based mutant library was screened by heat shock at 55°C for 1 minute. TdT3-1, identified as the top mutant from this library (Tables 4A and 4B), had a much higher FRET readout regardless of whether heat shock was applied or not, and retained a higher percentage of its FRET activity after heat shock (Figure 7C). Screening of the TdT1-3-based mutant library heat-treated at 58°C for 1 min revealed TdT3-2, which had a significantly higher FRET readout both at room temperature and after heat shock treatment (Figure 7D and Tables 4A and 4B). TdT3-2 retained at least half of its FRET activity after being subjected to 58°C for 1 min (Figure 7D). This suggests that TdT3-2 is significantly more active and thermostable than TdT1-3. As shown in Tables 4A and 4B, TdT3-2 carries the majority of the mutations identified from the top mutants from each round of screen, with the exception of T326S.

[0127] After identifying TdT3-2 as the optimal thermostable mutant, we optimized the expression and purification process to successfully obtain SUMO-TdT and TdT3-2 with minimal nuclease contamination (Figure 8). Because nucleases can interfere with downstream characterization studies, it is important that the purified SUMO-TdT and TdT3-2 have minimal nuclease activity.

[0128] Verification of the thermal stability of TdT3-2 Characterization of TdT3-2 thermal stability was performed by differential scanning calorimetry (DSC), differential scanning fluorimetry (DSF), SYPRO Orange thermal shift assay, and circular dichroism (CD). DSC measures the energy required to disrupt interactions that stabilize protein tertiary structure. The T obtained from DSC for SUMO-TdT m The T for SUMO-TdT and TdT3-2 from DSC was 40.2 °C and 50.7 °C for TdT3-2 (Figure 9A). m To further validate this, the same proteins were tested by DSF, which measures the fluorescence shift of endogenous tyrosine and tryptophan residues as the protein unfolds. The negative derivative of the fluorescence readings is plotted against temperature, and the maximum of this derivative curve corresponds to the T m SUMO-TdT corresponds to a T of 43.5°C. m was observed, but TdT3-2 had a T of 53.1°C. m (Figure 9B). m This was further validated by a SYPRO Orange thermal shift assay. SYPRO Orange dye binds nonspecifically to hydrophobic regions exposed during protein unfolding. The derivative of the change in fluorescence signal is the T m SUMO-TdT corresponds to a T of 41.5°C. m was recorded, whereas 51.5 °C was observed for TdT3-2 (Figure 9C). Circular dichroism (CD) is a spectroscopic technique for tracking protein unfolding as a function of temperature. CD measures characteristic spectral bands that can be attributed to α-helices and β-sheets. The change in CD as a function of temperature at characteristic wavelengths can be used to determine the midpoint of the protein unfolding transition (T m The first derivative value of the CD at a wavelength of 222 nm is plotted against temperature, and the maximum value is taken as T m The T of SUMO-TdT m is 45.7°C, and the T of TdT3-2 m was 52.5°C (Figure 9D).

[0129] T of both proteins m The CD measurements of the transition were slightly higher when compared to other techniques. The higher melting temperature may be related to the instrument settings. The sample cuvette was in contact with the Peltier holder via a metal adapter, which may result in a small heat dissipation and a small overestimation of the sample temperature by 1-2 °C. Nevertheless, the CD data were in good agreement with other experimental techniques, showing the same stability trends between the two proteins. The data confirmed that TdT3-2 was approximately 10 °C more thermostable than SUMO-TdT.

[0130] Primer extension reactions were performed using commercially available (NEB) TdT, SUMO-TdT, and TdT3-2 to determine whether TdT3-2 incorporates nucleotides at elevated temperatures. Reactions were performed at 25°C, 36°C, 47°C, and 58°C, and DNA products were visualized on a TBE-urea gel. All three enzymes were active at 25°C and 36°C (Figures 10A and 10B). At 47°C, commercially available TdT and SUMO-TdT denatured within 5 minutes, as the intensities of the incorporated bands remained the same for 5, 10, and 20 minutes. In contrast, TdT3-2 remained active for 20 minutes, as more incorporation was observed in the 20-minute reaction (Figure 10C). At 58°C, commercially available TdT and SUMO-TdT were not active, but TdT3-2 denatured within 5 minutes, as the band intensities remained the same thereafter (Figure 10D). This observation strongly confirms that TdT3-2 is more thermostable than SUMO-TdT and that TdT3-2 is active at higher temperatures.

[0131] TdT is used in library preparation to extend dsDNA samples prior to amplification. As TdT adds nucleotides to ssDNA, blunt-ended dsDNA relies on TdT to transiently melt DNA at the 3' end of the primer to incorporate the first few nucleotides. DNA samples with a high GC content have a higher T mThis can make it difficult to extend TdT. Increasing the temperature during library preparation using TdT would increase the proportion of dsDNA that transiently melts to ssDNA at the 3' end of the primer for TdT to incorporate nucleotides. However, the TdT used in library preparation is not thermostable. To demonstrate its potential in improving the library preparation process, we tested TdT3-2 on blunt-ended dsDNA containing four GC base pairs at the 3' end of the primer. Comparing the rate of ddCTP incorporation between commercially available TdT and TdT3-2, TdT3-2 performed better than commercially available TdT at both 37°C and 50°C. In addition, primer extension was higher for TdT3-2 at 50°C (Figures 11 and 12). This suggests the potential performance of TdT3-2 in improving current library preparation processes.

[0132] Kinetic characterization of SUMO-TdT and TdT3-2 To more precisely determine whether the thermostable TdT variants retained their terminal transferase activity, their steady-state activity was compared to that of SUMO-TdT. Steady-state assays were set up using excess DNA so that multiple turnovers could be observed, even if it was not possible to distinguish the number of incorporation events per enzyme turnover. Reactions at 37°C with titrations of dCTP concentration were quenched for 15 s each, and the amount of product was quantified by urea-PAGE to estimate the total turnover in micromolar concentrations of dCTP / s (see "Materials and Methods," Tables 5 and 6 for raw data). When this rate was plotted against dCTP concentration, a hyperbolic relationship consistent with Michaelis-Menten kinetics was observed, with a maximum elongation rate and a Michaelis constant (K). M TdT3-2 exhibited activity comparable to that of SUMO-TdT, with a slightly increased elongation rate and a lower K MThe results suggest that the enzyme has slightly improved catalytic activity (Table 7). Kinetic studies of SUMO-TdT and TdT3-2 at a higher temperature of 47°C were performed. The same analytical method was employed to obtain estimated total turnover in micromoles dCTP per second (Tables 8 and 9). TdT3-2 had comparable activity at both 37°C and 47°C, whereas SUMO-TdT had much lower activity at 47°C due to its inability to achieve saturation (Table 7).

[0133] [Table 6-1]

[0134] [Table 6-2]

[0135] [Table 7-1]

[0136] [Table 7-2]

[0137] [Table 8]

[0138] [Table 9]

[0139] [Table 10]

[0140] The affinity of TdT3-2 for the ssDNA oligo used in the extension reaction was estimated to be steady-state. Fluorescence polarization of the FAM-oligo in the presence of titrated TdT was measured, and the affinity for both our mutant and the SUMO-TdT enzyme was estimated to be greater than 1 μM (Figure 13). The thermostabilized mutant TdT3-2 retained the substrate binding affinity and catalytic properties of the SUMO-TdT enzyme, likely with a slight improvement in activity.

[0141] Homology-based modeling of TdT3-2 The mouse TdT crystal structure was available (PDB ID 4I27). Because TdT3-2 is derived from a bovine source, visualizing the location of mutations on the 3D structure could only be done by homology modeling. Based on the available homologous sequences and known structures, the Phyre2 server generated a 3D structure of TdT3-2 from its amino acid query sequence. The locations of the eight mutations and other important residues could be visualized in the model obtained from Phyre2 (Figure 14). However, this model could not accurately account for the effects of the acquired mutations in TdT3-2 that contribute to its improved thermostability.

[0142] conclusion Thermostable TdT variants may be useful for practical applications, such as minimizing the presence of secondary structures during oligonucleotide synthesis by increasing the temperature. Thermostable variants of mouse TdT were able to improve the extension rate of several GC-rich hairpin primers by increasing the temperature during the incorporation reaction. The protein engineering method used to discover TdT3-2 in this example enabled the exploration of a broad sequence space. This example demonstrates the value and significance of evolving thermostable TdT for applications in DNA synthesis.

[0143] This example establishes a adaptable plate-based FRET assay for screening mutant libraries using E. coli cell lysates. A third round of screening led to the discovery of a significantly more thermostable variant, TdT3-2. TdT3-2 exhibited a T ~10°C higher than SUMO-TdT, as verified by DSC, DSF, SYPRO Orange thermal shift assay, and CD. m Additionally, the improved thermostability did not come at the expense of enzymatic activity. TdT3-2 is a better tool for the wide range of applications currently using SUMO-TdT due to its improved thermostability and robust activity. Furthermore, TdT3-2 is also an ideal starting point for further engineering to ultimately evolve a TdT that can efficiently incorporate reversibly blocked nucleotides, which would be particularly useful in the emerging fields of de novo gene synthesis and information storage in DNA.

[0144] Together, these data demonstrate that the nine amino acid substitutions identified in this example, individually or in any combination, can increase the thermostability of TdT mutants containing the amino acid substitution(s) while preserving TdT catalytic activity. Furthermore, the TdT mutants identified in this example can be thermostable while maintaining TdT catalytic activity.

[0145] term In at least some of the foregoing embodiments, one or more elements used in one embodiment may be used interchangeably in another embodiment, except where such an exchange is not technically feasible. Those skilled in the art will appreciate that various other omissions, additions, and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and variations are intended to be included within the scope of the subject matter, as defined by the appended claims.

[0146] For the use of virtually any plural and / or singular term herein, those of ordinary skill in the art can substitute plural for singular and / or singular for plural as appropriate to the context and / or application. For clarity, various singular / plural permutations may be expressly set forth herein. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Any reference herein to "or" is intended to encompass "and / or" unless specifically indicated otherwise.

[0147] It will be understood by those skilled in the art that, in general, the terminology used herein, and particularly in the appended claims (e.g., the body of the appended claims), is generally intended as "open" terms (e.g., the term "including" should be interpreted as "including but not limited to," the term "having" should be interpreted as "having at least," the term "includes" should be interpreted as "includes but is not limited to," etc.). It will also be understood by those skilled in the art that where a specific number of introduced claim recitations is intended, such intention will be expressly recited in the claim; in the absence of such recitation, no such intention exists. For example, to aid in understanding, the following appended claims may include the use of the introductory phrases "at least one" and "one or more" to introduce claim recitations. However, the use of such phrases does not mean that the introduction of a claim recitation with the indefinite article "a" or "an" should be construed as limiting any particular claim containing such an introduced claim recitation to embodiments containing only one of such recitations, even when the same claim also includes "one or more" or "at least one" and an indefinite article such as "a" or "an" (e.g., "a" and / or "an" should be interpreted to mean "at least one" or "one or more"), and the same applies to the use of indefinite articles used to introduce claim recitations. Furthermore, even when a specific number of introduced claim recitations is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the unmodified recitation of "two recitations" means at least two recitations or more than two recitations, in the absence of other modifications).Furthermore, when phrases similar to "at least one of A, B, and C, etc." are used, generally such structures are intended in the sense that one of ordinary skill in the art would understand the phrase (e.g., "a system having at least one of A, B, and C" includes, but is not limited to, systems having A only, B only, C only, both A and B, both A and C, both B and C, and / or both A, B, and C, etc.). When phrases similar to "at least one of A, B, or C, etc." are used, generally such structures are intended in the sense that one of ordinary skill in the art would understand the phrase (e.g., "a system having at least one of A, B, or C" includes, but is not limited to, systems having A only, B only, C only, both A and B, both A and C, both B and C, and / or both A, B, and C, etc.). Those skilled in the art will further appreciate that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the specification, claims, or drawings, should be understood to contemplate the possibility of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B."

[0148] Additionally, where features or aspects of the disclosure are described in terms of a Markush group, one skilled in the art will thereby recognize that the disclosure is also described in terms of any individual members or subgroups of members of the Markush group.

[0149] As will be understood by those skilled in the art, for any and all purposes, including in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of those subranges. Any recited range can be readily recognized as fully descriptive and allowing for the same range to be broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range described herein can be readily broken down into a lower third, middle third, upper third, etc. As will also be understood by those skilled in the art, all language, such as "up to," "at least," "greater than," and "less than," refers to a range that is inclusive of the recited numbers and can subsequently be broken down into subranges as described above. Finally, as will be understood by those skilled in the art, a range includes each individual component. Thus, for example, a group having 1 to 3 items means a group having 1, 2, or 3 items. Similarly, a group having 1 to 5 items means a group having 1, 2, 3, 4, or 5 items, etc.

[0150] Aspects of the present invention are further described in the following sections: [Section 1] 1. A recombinant terminal deoxynucleotidyl transferase (TdT) comprising an amino acid sequence that is at least 80% identical to SEQ ID NO: 1, wherein the recombinant TdT comprises one or more amino acid substitution mutations at one or more positions functionally equivalent to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 of Bos taurus TdT of SEQ ID NO: 12. [Section 2] The recombinant TdT described in item 1 above, wherein the amino acid substitution mutation at a position functionally equivalent to Glu191 of Bos taurus TdT of SEQ ID NO: 12 includes a mutation to a nonpolar amino acid, a polar amino acid, a positively charged amino acid, a negatively charged amino acid, a hydrophobic amino acid, an aromatic amino acid, an aliphatic amino acid, a small amino acid, or a hydrophilic amino acid. [Section 3] The recombinant TdT according to item 1 above, wherein the amino acid substitution mutation at a position functionally equivalent to Glu191 of Bos taurus TdT of SEQ ID NO: 12 is Glu191Ala, Glu191Arg, Glu191Asn, Glu191Asp, Glu191Cys, Glu191Gln, Glu191Gly, Glu191His, Glu191Ile, Glu191Leu, Glu191Lys, Glu191Met, Glu191Phe, Glu191Pro, Glu191Ser, Glu191Thr, Glu191Trp, Glu191Tyr, or Glu191Val. [Section 4] Item 1. The recombinant TdT according to Item 1, wherein the amino acid substitution mutation at a position functionally equivalent to Glu191 of Bos taurus TdT of SEQ ID NO: 12 is Glu191Val. [Section 5] 5. The recombinant TdT according to any one of items 1 to 4, wherein the amino acid substitution mutation at a position functionally equivalent to Lys193 of Bos taurus TdT of SEQ ID NO: 12 comprises a mutation to a nonpolar amino acid, a polar amino acid, a positively charged amino acid, a negatively charged amino acid, a hydrophobic amino acid, an aromatic amino acid, an aliphatic amino acid, a small amino acid, or a hydrophilic amino acid. [Section 6] 5. The recombinant TdT according to any one of items 1 to 4, wherein the amino acid substitution mutation at a position functionally equivalent to Lys193 of Bos taurus TdT of SEQ ID NO: 12 is Lys193Ala, Lys193Arg, Lys193Asn, Lys193Asp, Lys193Cys, Lys193Gln, Lys193Glu, Lys193Gly, Lys193His, Lys193Ile, Lys193Leu, Lys193Met, Lys193Phe, Lys193Pro, Lys193Ser, Lys193Thr, Lys193Trp, Lys193Tyr, or Lys193Val. [Section 7] 5. The recombinant TdT according to any one of items 1 to 4, wherein the amino acid substitution mutation at a position functionally equivalent to Lys193 of Bos taurus TdT of SEQ ID NO: 12 is Lys193Asn. [Section 8] 8. The recombinant TdT according to any one of items 1 to 7, wherein the amino acid substitution mutation at a position functionally equivalent to Glu194 of Bos taurus TdT of SEQ ID NO: 12 comprises a mutation to a nonpolar amino acid, a polar amino acid, a positively charged amino acid, a negatively charged amino acid, a hydrophobic amino acid, an aromatic amino acid, an aliphatic amino acid, a small amino acid, or a hydrophilic amino acid. [Section 9] 8. The recombinant TdT according to any one of items 1 to 7, wherein the amino acid substitution mutation at a position functionally equivalent to Glu194 of Bos taurus TdT of SEQ ID NO: 12 is Glu194Ala, Glu194Arg, Glu194Asn, Glu194Asp, Glu194Cys, Glu194Gln, Glu194Gly, Glu194His, Glu194Ile, Glu194Leu, Glu194Lys, Glu194Met, Glu194Phe, Glu194Pro, Glu194Ser, Glu194Thr, Glu194Trp, Glu194Tyr, or Glu194Val. [Section 10] 8. The recombinant TdT according to any one of items 1 to 7, wherein the amino acid substitution mutation at a position functionally equivalent to Glu194 of Bos taurus TdT of SEQ ID NO: 12 is Glu194Gly. [Section 11] 11. The recombinant TdT according to any one of items 1 to 10, wherein the amino acid substitution mutation at a position functionally equivalent to Asp242 of Bos taurus TdT of SEQ ID NO: 12 comprises a mutation to a nonpolar amino acid, a polar amino acid, a positively charged amino acid, a negatively charged amino acid, a hydrophobic amino acid, an aromatic amino acid, an aliphatic amino acid, a small amino acid, or a hydrophilic amino acid. [Section 12] 11. The recombinant TdT according to any one of items 1 to 10, wherein the amino acid substitution mutation at a position functionally equivalent to Asp242 of Bos taurus TdT of SEQ ID NO: 12 is Asp242Ala, Asp242Arg, Asp242Asn, Asp242Cys, Asp242Gln, Asp242Glu, Asp242Gly, Asp242His, Asp242Ile, Asp242Leu, Asp242Lys, Asp242Met, Asp242Phe, Asp242Pro, Asp242Ser, Asp242Thr, Asp242Trp, Asp242Tyr, or Asp242Val. [Section 13] 11. The recombinant TdT according to any one of items 1 to 10, wherein the amino acid substitution mutation at a position functionally equivalent to Asp242 of Bos taurus TdT of SEQ ID NO: 12 is Asp242Tyr. [Section 14] 14. The recombinant TdT according to any one of items 1 to 13, wherein the amino acid substitution mutation at a position functionally equivalent to Lys287 of Bos taurus TdT of SEQ ID NO: 12 comprises a mutation to a nonpolar amino acid, a polar amino acid, a positively charged amino acid, a negatively charged amino acid, a hydrophobic amino acid, an aromatic amino acid, an aliphatic amino acid, a small amino acid, or a hydrophilic amino acid. [Section 15] 14. The recombinant TdT according to any one of items 1 to 13, wherein the amino acid substitution mutation at a position functionally equivalent to Lys287 of Bos taurus TdT of SEQ ID NO: 12 is Lys287Ala, Lys287Arg, Lys287Asn, Lys287Asp, Lys287Cys, Lys287Gln, Lys287Glu, Lys287Gly, Lys287His, Lys287Ile, Lys287Leu, Lys287Met, Lys287Phe, Lys287Pro, Lys287Ser, Lys287Thr, Lys287Trp, Lys287Tyr, or Lys287Val. [Section 16] 14. The recombinant TdT according to any one of items 1 to 13, wherein the amino acid substitution mutation at a position functionally equivalent to Lys287 of Bos taurus TdT of SEQ ID NO: 12 is Lys287Glu. [Section 17] 17. The recombinant TdT according to any one of items 1 to 16, wherein the amino acid substitution mutation at a position functionally equivalent to Phe296 of Bos taurus TdT of SEQ ID NO: 12 comprises a mutation to a nonpolar amino acid, a polar amino acid, a positively charged amino acid, a negatively charged amino acid, a hydrophobic amino acid, an aromatic amino acid, an aliphatic amino acid, a small amino acid, or a hydrophilic amino acid. [Section 18] 17. The recombinant TdT according to any one of items 1 to 16, wherein the amino acid substitution mutation at a position functionally equivalent to Phe296 of Bos taurus TdT of SEQ ID NO: 12 is Phe296Ala, Phe296Arg, Phe296Asn, Phe296Asp, Phe296Cys, Phe296Gln, Phe296Glu, Phe296Gly, Phe296His, Phe296Ile, Phe296Leu, Phe296Lys, Phe296Met, Phe296Pro, Phe296Ser, Phe296Thr, Phe296Trp, Phe296Tyr, or Phe296Val. [Section 19] 17. The recombinant TdT according to any one of items 1 to 16, wherein the amino acid substitution mutation at a position functionally equivalent to Phe296 of Bos taurus TdT of SEQ ID NO: 12 is Phe296Leu. [Section 20] 20. The recombinant TdT according to any one of items 1 to 19, wherein the amino acid substitution mutation at a position functionally equivalent to Met299 of Bos taurus TdT of SEQ ID NO: 12 comprises a mutation to a nonpolar amino acid, a polar amino acid, a positively charged amino acid, a negatively charged amino acid, a hydrophobic amino acid, an aromatic amino acid, an aliphatic amino acid, a small amino acid, or a hydrophilic amino acid. [Section 21] 20. The recombinant TdT according to any one of items 1 to 19, wherein the amino acid substitution mutation at a position functionally equivalent to Met299 of Bos taurus TdT of SEQ ID NO: 12 is Met299Ala, Met299Arg, Met299Asn, Met299Asp, Met299Cys, Met299Gln, Met299Glu, Met299Gly, Met299His, Met299Ile, Met299Leu, Met299Lys, Met299Phe, Met299Pro, Met299Ser, Met299Thr, Met299Trp, Met299Tyr, or Met299Val. [Section 22] 20. The recombinant TdT according to any one of items 1 to 19, wherein the amino acid substitution mutation at a position functionally equivalent to Met299 of Bos taurus TdT of SEQ ID NO: 12 is Met299Lys. [Section 23] 23. The recombinant TdT according to any one of items 1 to 22, wherein the amino acid substitution mutation at a position functionally equivalent to Thr342 of Bos taurus TdT of SEQ ID NO: 12 comprises a mutation to a nonpolar amino acid, a polar amino acid, a positively charged amino acid, a negatively charged amino acid, a hydrophobic amino acid, an aromatic amino acid, an aliphatic amino acid, a small amino acid, or a hydrophilic amino acid. [Section 24] 23. The recombinant TdT according to any one of items 1 to 22, wherein the amino acid substitution mutation at a position functionally equivalent to Thr342 of Bos taurus TdT of SEQ ID NO: 12 is Thr342Ala, Thr342Arg, Thr342Asn, Thr342Asp, Thr342Cys, Thr342Gln, Thr342Glu, Thr342Gly, Thr342His, Thr342Ile, Thr342Leu, Thr342Lys, Thr342Met, Thr342Phe, Thr342Pro, Thr342Ser, Thr342Trp, Thr342Tyr, or Thr342Val. [Section 25] 23. The recombinant TdT according to any one of items 1 to 22, wherein the amino acid substitution mutation at a position functionally equivalent to Thr342 of Bos taurus TdT of SEQ ID NO: 12 is Thr342Ser. [Section 26] 26. The recombinant TdT according to any one of items 1 to 25, wherein the amino acid substitution mutation at a position functionally equivalent to His421 of Bos taurus TdT of SEQ ID NO: 12 comprises a mutation to a nonpolar amino acid, a polar amino acid, a positively charged amino acid, a negatively charged amino acid, a hydrophobic amino acid, an aromatic amino acid, an aliphatic amino acid, a small amino acid, or a hydrophilic amino acid. [Section 27] 26. The recombinant TdT according to any one of items 1 to 25, wherein the amino acid substitution mutation at a position functionally equivalent to His421 of Bos taurus TdT of SEQ ID NO: 12 is His421Ala, His421Arg, His421Asn, His421Asp, His421Cys, His421Gln, His421Glu, His421Gly, His421Ile, His421Leu, His421Lys, His421Met, His421Phe, His421Pro, His421Ser, His421Thr, His421Trp, His421Tyr, or His421Val. [Section 28] 26. The recombinant TdT according to any one of items 1 to 25, wherein the amino acid substitution mutation at a position functionally equivalent to His421 of Bos taurus TdT of SEQ ID NO: 12 is His421Pro. [Section 29] 29. The recombinant TdT according to any one of items 1 to 28, wherein the recombinant TdT comprises two or more amino acid substitution mutations at two or more positions functionally equivalent to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 of Bos taurus TdT of SEQ ID NO: 12. [Section 30] 30. The method of claim 29, wherein the two or more amino acid substitution mutations at two or more positions functionally equivalent to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 of Bos taurus TdT of SEQ ID NO: 12 include two or more of Glu191Val, Lys193Asn, Glu194Gly, Asp242Tyr, Lys287Glu, Phe296Leu, Met299Lys, Thr342Ser, and His421Pro, respectively. [Section 31] 29. The recombinant TdT according to any one of Items 1 to 28, wherein the recombinant TdT comprises three or more amino acid substitution mutations at three or more positions functionally equivalent to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 of Bos taurus TdT of SEQ ID NO: 12. [Section 32] 32. The method according to claim 31, wherein the three or more amino acid substitution mutations at three or more positions functionally equivalent to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 of Bos taurus TdT of SEQ ID NO: 12 include three or more of Glu191Val, Lys193Asn, Glu194Gly, Asp242Tyr, Lys287Glu, Phe296Leu, Met299Lys, Thr342Ser, and His421Pro, respectively. [Section 33] 29. The recombinant TdT according to any one of Items 1 to 28, wherein the recombinant TdT comprises four or more amino acid substitution mutations at four or more positions functionally equivalent to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 of Bos taurus TdT of SEQ ID NO: 12. [Section 34] 34. The method according to claim 33, wherein the four or more amino acid substitution mutations at four or more positions functionally equivalent to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 of Bos taurus TdT of SEQ ID NO: 12 include four or more of Glu191Val, Lys193Asn, Glu194Gly, Asp242Tyr, Lys287Glu, Phe296Leu, Met299Lys, Thr342Ser, and His421Pro, respectively. [Section 35] 29. The recombinant TdT according to any one of Items 1 to 28, wherein the recombinant TdT comprises five or more amino acid substitution mutations at five or more positions functionally equivalent to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 of Bos taurus TdT of SEQ ID NO: 12. [Section 36] 36. The method according to item 35, wherein the five or more amino acid substitution mutations at five or more positions functionally equivalent to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 of Bos taurus TdT of SEQ ID NO: 12 include five or more of Glu191Val, Lys193Asn, Glu194Gly, Asp242Tyr, Lys287Glu, Phe296Leu, Met299Lys, Thr342Ser, and His421Pro, respectively. [Section 37] 29. The recombinant TdT according to any one of Items 1 to 28, wherein the recombinant TdT comprises six or more amino acid substitution mutations at six or more positions functionally equivalent to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 of Bos taurus TdT of SEQ ID NO: 12. [Section 38] 38. The method according to item 37, wherein the six or more amino acid substitution mutations at six or more positions functionally equivalent to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 of Bos taurus TdT of SEQ ID NO: 12 include six or more of Glu191Val, Lys193Asn, Glu194Gly, Asp242Tyr, Lys287Glu, Phe296Leu, Met299Lys, Thr342Ser, and His421Pro, respectively. [Section 39] 29. The recombinant TdT according to any one of Items 1 to 28, wherein the recombinant TdT comprises seven or more amino acid substitution mutations at seven or more positions functionally equivalent to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 of Bos taurus TdT of SEQ ID NO: 12. [Section 40] 40. The method according to item 39, wherein the seven or more amino acid substitution mutations at seven or more positions functionally equivalent to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 of Bos taurus TdT of SEQ ID NO: 12 include seven or more of Glu191Val, Lys193Asn, Glu194Gly, Asp242Tyr, Lys287Glu, Phe296Leu, Met299Lys, Thr342Ser, and His421Pro, respectively. [Section 41] 29. The recombinant TdT according to any one of Items 1 to 28, wherein the recombinant TdT comprises eight or more amino acid substitution mutations at eight or more positions functionally equivalent to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 of Bos taurus TdT of SEQ ID NO: 12. [Section 42] 42. The method according to item 41, wherein the eight or more amino acid substitution mutations at eight or more positions functionally equivalent to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 of Bos taurus TdT of SEQ ID NO: 12 include eight or more of Glu191Val, Lys193Asn, Glu194Gly, Asp242Tyr, Lys287Glu, Phe296Leu, Met299Lys, Thr342Ser, and His421Pro, respectively. [Section 43] 29. The recombinant TdT according to any one of items 1 to 28, wherein the recombinant TdT comprises eight amino acid substitution mutations at eight positions functionally equivalent to Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 of Bos taurus TdT of SEQ ID NO: 12. [Section 44] 44. The method according to item 43, wherein the eight amino acid substitution mutations at eight positions functionally equivalent to Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 of Bos taurus TdT of SEQ ID NO: 12 are Lys193Asn, Glu194Gly, Asp242Tyr, Lys287Glu, Phe296Leu, Met299Lys, Thr342Ser, and His421Pro, respectively. [Section 45] 29. The recombinant TdT according to any one of items 1 to 28, wherein the recombinant TdT comprises nine amino acid substitution mutations at positions functionally equivalent to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 of Bos taurus TdT of SEQ ID NO: 12. [Section 46] 46. ​​The method according to item 45, wherein the nine amino acid substitution mutations at positions functionally equivalent to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 of Bos taurus TdT of SEQ ID NO: 12 are Glu191Val, Lys193Asn, Glu194Gly, Asp242Tyr, Lys287Glu, Phe296Leu, Met299Lys, Thr342Ser, and His421Pro, respectively. [Section 47] 46. ​​The recombinant TdT according to any one of items 1 to 45, wherein the recombinant TdT comprises an amino acid sequence that is at least 85% identical to SEQ ID NO:1. [Section 48] 46. ​​The recombinant TdT according to any one of items 1 to 45, wherein the recombinant TdT comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:1. [Section 49] 46. ​​The recombinant TdT according to any one of items 1 to 45, wherein the recombinant TdT comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:1. [Section 50] 46. ​​The recombinant TdT according to any one of items 1 to 45, wherein the recombinant TdT comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:11. [Section 51] 46. ​​The recombinant TdT according to any one of items 1 to 45, wherein the recombinant TdT comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:12. [Section 52] 52. The recombinant TdT according to any one of items 1 to 51, wherein the recombinant TdT is stable at a temperature of 47° C. or higher. [Section 53] 52. The recombinant TdT according to any one of items 1 to 51, wherein the recombinant TdT is stable at a temperature of 50°C or higher. [Section 54] 52. The recombinant TdT according to any one of items 1 to 51, wherein the recombinant TdT is stable at a temperature of 55°C or higher. [Section 55] 52. The recombinant TdT according to any one of items 1 to 51, wherein the recombinant TdT is stable at a temperature of 58°C or higher. [Section 56] 56. The recombinant TdT according to any one of items 1 to 55, wherein the terminal deoxynucleotidyl transferase activity of the recombinant TdT is at least 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, or 120% of the terminal deoxynucleotidyl transferase activity of Bos taurus TdT of SEQ ID NO: 12 at the same test temperature. [Section 57] 57. The recombinant TdT according to item 56, wherein the test temperature is 37°C, 47°C, 50°C, 55°C, or 58°C. [Section 58] 58. The recombinant TdT according to any one of items 1 to 57, wherein the recombinant TdT comprises a small ubiquitin-like modifier (SUMO) fragment. [Section 59] 59. The recombinant TdT of paragraph 58, wherein the SUMO fragment comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:13. [Section 60] 60. The recombinant TdT according to any one of items 58 to 59, wherein the recombinant TdT comprises a SUMO fragment on the N-terminus of the recombinant TdT. [Section 61] 60. The recombinant TdT of paragraph 59, wherein the recombinant TdT comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:14. [Section 62] 60. The recombinant TdT of paragraph 59, wherein the recombinant TdT comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:15. [Section 63] 60. The recombinant TdT according to any one of items 58 to 59, wherein the recombinant TdT comprises a SUMO fragment on the C-terminus of the recombinant TdT. [Section 64] 64. A polynucleotide encoding the recombinant terminal deoxynucleotidyl transferase (TdT) according to any one of items 1 to 63. [Section 65] 65. An expression vector comprising the polynucleotide according to Item 64. [Section 66] 64. The recombinant terminal deoxynucleotidyl transferase (TdT) according to any one of Items 1 to 63, 65. The polynucleotide according to Item 64, Item 65. The expression vector according to item 65, A host cell comprising one or more of: [Section 67] A kit comprising: A recombinant terminal deoxynucleotidyl transferase (TdT) according to any one of Items 1 to 63, a polynucleotide according to Item 64, an expression vector according to Item 65, a host cell according to Item 66, or a combination thereof; and instructions for using the recombinant terminal deoxynucleotidyl transferase, the polynucleotide, the expression vector, the host cell, or a combination thereof. While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are illustrative and not intended to be limiting, with the true scope and spirit being indicated by the following claims.

Claims

1. A recombinant terminal deoxynucleotidyl transferase (TdT) comprising an amino acid sequence at least 90% identical to SEQ ID NO:1, wherein the recombinant TdT comprises one or more amino acid substitution mutations at one or more positions corresponding to Glu191, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 of Bos taurus TdT of SEQ ID NO:12; the amino acid substitution mutation at the position corresponding to Glu191 of Bos taurus TdT in SEQ ID NO: 12 is Glu191Val; the amino acid substitution mutation at the position corresponding to Glu194 of Bos taurus TdT in SEQ ID NO: 12 is Glu194Gly; the amino acid substitution mutation at the position corresponding to Asp242 of Bos taurus TdT in SEQ ID NO: 12 is Asp242Tyr; the amino acid substitution mutation at the position corresponding to Lys287 of Bos taurus TdT in SEQ ID NO: 12 is Lys287Glu; the amino acid substitution mutation at the position corresponding to Phe296 of Bos taurus TdT in SEQ ID NO: 12 is Phe296Leu; the amino acid substitution mutation at the position corresponding to Met299 of Bos taurus TdT in SEQ ID NO: 12 is Met299Lys; the amino acid substitution mutation at the position corresponding to Thr342 of Bos taurus TdT in SEQ ID NO: 12 is Thr342Ser; the amino acid substitution mutation at the position corresponding to His421 of Bos taurus TdT in SEQ ID NO: 12 is His421Pro; Recombinant terminal deoxynucleotidyl transferase (TdT), wherein the recombinant TdT has improved thermostability.

2. 2. The recombinant TdT of claim 1, wherein the recombinant TdT comprises two or more amino acid substitution mutations at two or more positions corresponding to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 of Bos taurus TdT of SEQ ID NO: 12, and the amino acid substitution mutation at the position corresponding to Lys193 of Bos taurus TdT of SEQ ID NO: 12 is Lys193Asn.

3. The recombinant TdT of claim 1 or 2, wherein the recombinant TdT comprises nine amino acid substitution mutations at positions corresponding to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 of Bos taurus TdT of SEQ ID NO: 12, and the amino acid substitution mutation at the position corresponding to Lys193 of Bos taurus TdT of SEQ ID NO: 12 is Lys193Asn.

4. The recombinant TdT according to any one of claims 1 to 3, wherein the recombinant TdT comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:

11.

5. The recombinant TdT according to any one of claims 1 to 4, wherein the recombinant TdT is stable at a temperature of 47°C or higher.

6. The recombinant TdT of any one of claims 1 to 5, wherein the terminal deoxynucleotidyl transferase activity of the recombinant TdT is at least 80% of the terminal deoxynucleotidyl transferase activity of Bos taurus TdT of SEQ ID NO: 12 at the same test temperature.

7. The recombinant TdT of any one of claims 1 to 6, wherein the recombinant TdT comprises a small ubiquitin-like modifier (SUMO) fragment, and optionally the recombinant TdT comprises a SUMO fragment on the N-terminus of the recombinant TdT.

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