Base-modified nucleotides as substrates for tdt-based enzymatic nucleic acid synthesis
US-2022025421-A1 · Jan 27, 2022 · US
Thermostable terminal deoxynucleotidyl transferase
US12110516B2 · US · B2
| Field | Value |
|---|---|
| Publication number | US-12110516-B2 |
| Application number | US-202117317721-A |
| Country | US |
| Kind code | B2 |
| Filing date | May 11, 2021 |
| Priority date | May 12, 2020 |
| Publication date | Oct 8, 2024 |
| Grant date | Oct 8, 2024 |
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- Title
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Abstract
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Disclosed herein include recombinant terminal deoxynucleotidyl transferases (TdTs). In some embodiments, the recombinant TdT comprises an amino acid sequence that is at least 80% identical to a 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 in the bovine TdT.
First claim
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What is claimed is: 1. A recombinant terminal deoxynucleotidyl transferase (TdT) comprising an amino acid sequence that is 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, Met299, Thr342, and His421 in the Bos taurus TdT of SEQ ID NO: 12. 2. The recombinant TdT of claim 1 , wherein the amino acid substitution mutation at the position corresponding to Glu191 in the Bos taurus TdT of SEQ ID NO: 12 is Glu191Val. 3. The recombinant TdT of claim 1 , wherein the amino acid substitution mutation at the position corresponding to Glu194 in the Bos taurus TdT of SEQ ID NO: 12 is Glu194Gly. 4. The recombinant TdT of claim 1 , wherein the amino acid substitution mutation at the position corresponding to Asp242 in the Bos taurus TdT of SEQ ID NO: 12 is Asp242Tyr. 5. The recombinant TdT of claim 1 , wherein the amino acid substitution mutation at the position corresponding to Lys287 in the Bos taurus TdT of SEQ ID NO: 12 is Lys287Glu. 6. The recombinant TdT of claim 1 , wherein the amino acid substitution mutation at the position corresponding to Met299 in the Bos taurus TdT of SEQ ID NO: 12 is Met299Lys. 7. The recombinant TdT of claim 1 , wherein the amino acid substitution mutation at the position corresponding to Thr342 in the Bos taurus TdT of SEQ ID NO: 12 is Thr342Ser. 8. The recombinant TdT of claim 1 , wherein the amino acid substitution mutation at the position corresponding to His421 in the Bos taurus TdT of SEQ ID NO: 12 is His421Pro. 9. 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, Met299, Thr342, and His421 in the Bos taurus TdT of SEQ ID NO: 12. 10. The recombinant TdT of claim 1 , wherein the recombinant TdT comprises nine amino acid substitution mutations at positions corresponding to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 in the Bos taurus TdT of SEQ ID NO: 12. 11. The recombinant TdT of claim 1 , wherein the recombinant TdT comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 11. 12. The recombinant TdT of claim 1 , wherein the recombinant TdT comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 12. 13. The recombinant TdT of claim 1 , wherein the recombinant TdT is stable at a temperature of 47° C.or higher. 14. The recombinant TdT of claim 1 , 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 the Bos taurus TdT of SEQ ID NO: 12 at a same test temperature. 15. The recombinant TdT of claim 1 , wherein the recombinant TdT comprises a small ubiquitin-like modifier (SUMO) fragment. 16. The recombinant TdT of claim 1 , wherein the recombinant TdT comprises the SUMO fragment on the N-terminus of the recombinant TdT. 17. The recombinant TdT of claim 1 , wherein the recombinant TdT comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 1. 18. The recombinant TdT of claim 1 , wherein the recombinant TdT comprises an amino acid sequence that is at least 99% identical to SEQ ID NO: 1. 19. The recombinant TdT of claim 1 , wherein the recombinant TdT comprises three or more amino acid substitution mutations at three or more positions corresponding to Glu191, Lys193, Glu194, Asp242, Lys287, Phe296, Met299, Thr342, and His421 in the Bos taurus TdT of SEQ ID NO: 12. 20. The recombinant TdT of claim 1 , wherein the recombinant TdT comprises an amino acid substitution mutation at a position corresponding to Lys193 in the Bos taurus TdT of SEQ ID NO: 12. 21. The recombinant TdT of claim 20 , wherein the amino acid substitution mutation at the position corresponding to Lys193 in the Bos taurus TdT of SEQ ID NO: 12 is Lys193Asn. 22. The recombinant TdT of claim 1 , wherein the recombinant TdT comprises an amino acid substitution mutation at a position corresponding to Phe296 in the Bos taurus TdT of SEQ ID NO: 12. 23. The recombinant TdT of claim 22 , wherein the amino acid substitution mutation at the position corresponding to Phe296 in the Bos taurus TdT of SEQ ID NO: 12 is Phe296Leu.
Assignees
Inventors
Classifications
DNA nucleotidylexotransferase (2.7.7.31), i.e. terminal deoxynucleotidyl transferase · CPC title
containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin · CPC title
- C12N9/1264Primary
DNA nucleotidylexotransferase (2.7.7.31), i.e. terminal nucleotidyl transferase · CPC title
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- What does patent US12110516B2 cover?
- Disclosed herein include recombinant terminal deoxynucleotidyl transferases (TdTs). In some embodiments, the recombinant TdT comprises an amino acid sequence that is at least 80% identical to a 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,…
- Who is the assignee on this patent?
- Illumina Inc, Illumina Singapore Pte Ltd, Nat Univ Singapore
- What technology area does this patent fall under?
- Primary CPC classification C12N9/1264. Mapped technology areas include Chemistry & Metallurgy.
- When was this patent published?
- Publication date Tue Oct 08 2024 00:00:00 GMT+0000 (Coordinated Universal Time) (B2). Legal status and post-grant events are not shown on this page.
- What related patents are in patentsdb?
- We list 3 related publications on this page (citations in our corpus or others sharing the same primary CPC).