Modified biotin-binding proteins for immobilization

• US 10,655,168 B2
• Filed: 12/21/2018
• Issued: 05/19/2020
• Est. Priority Date: 12/22/2017
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Kamtekar, Satwik, Sebo, Lubomir, Chern, Leewin, Linsky, Thomas, Hanes, Jeremiah, Miller, Erik, Yang, Ying, Yue, Stephen
• Primary Examiner(s): Steele, Amber D
• Application Number:
  US16/230,480
• Publication Number:
  US 20190249237A1
• Time in Patent Office: 515 Days
• CPC Class Codes: C07K 14/36 from Actinomyces; from Streptomyces (G)
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 2535/00 Reactions characterised by the assay type for determining the identity of a nucleotide base or a sequence of oligonucleotides
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis

Abstract

Compositions comprising covalently modified and mutated biotin-binding proteins, particularly biotin-binding proteins having a negative charge at physiological pH, are provided. Methods of producing such proteins are also provided, as are methods of immobilizing, sequencing, and making nucleic acids employing such proteins.

Claims

1. A composition comprising a modified biotin-binding protein that comprises one or more covalently attached sulfonate moieties.

2. The composition of claim 1, wherein the biotin-binding protein is a tetravalent biotin-binding protein.

3. The composition of claim 1, wherein the biotin-binding protein is streptavidin.

4. The composition of claim 1, wherein the biotin-binding protein comprises three or more covalently attached sulfonate moieties.

5. The composition of claim 1, wherein the biotin-binding protein comprises 12 or more covalently attached sulfonate moieties.

6. The composition of claim 1, wherein the biotin-binding protein comprises 45 or more covalently attached sulfonate moieties.

7. The composition of claim 1, wherein the biotin-binding protein comprises one or more covalently attached 3,4,5-tris(3-sulfopropoxy)benzoyl moieties.

8. The composition of claim 1, wherein the biotin-binding protein comprises 15 or more covalently attached 3,4,5-tris(3-sulfopropoxy)benzoyl moieties.

9. The composition of claim 1, wherein the biotin-binding protein is a tetravalent biotin-binding protein comprising 45 or more covalently attached sulfonate moieties.

10. The composition of claim 9, wherein the biotin-binding protein is streptavidin.

11. The composition of claim 1, wherein the biotin-binding protein is a tetravalent biotin-binding protein comprising 15 or more covalently attached 3,4,5-tris(3-sulfopropoxy)benzoyl moieties.

12. The composition of claim 11, wherein the biotin-binding protein is streptavidin.

13. The composition of claim 1, wherein the biotin-binding protein comprises one or more covalently attached 3,5-disulfobenzoyl or 2-sulfobenzoyl moieties.

14. The composition of claim 1, wherein the biotin-binding protein comprises one or more covalently attached polyethylene glycol (PEG) moieties.

15. The composition of claim 1, wherein the biotin-binding protein comprises one or more amino acid substitutions that decrease its calculated net charge relative to a parental biotin-binding protein.

16. The composition of claim 1, wherein the biotin-binding protein is bound to a nucleic acid polymerase.

17. The composition of claim 16, wherein the nucleic acid polymerase is complexed with a nucleic acid.

18. The composition of claim 1, wherein the biotin-binding protein is immobilized on a solid support.

19. The composition of claim 1, wherein the biotin-binding protein is immobilized on the base of a nanoscale well.

20. The composition of claim 1, wherein the composition is present in a nucleic acid sequencing system.

22. The composition of claim 1, wherein the biotin-binding protein is a tetrameric biotin-binding protein.

23. The composition of claim 1, wherein the biotin-binding protein is traptavidin.

21. A system for sequencing nucleic acids, the system comprising

a chip comprising a plurality of polymerase enzyme complexes bound thereto, each polymerase enzyme complex individually optically resolvable, each polymerase enzyme complex comprising a polymerase enzyme, a template nucleic acid, and optionally a primer hybridized to the template nucleic acid, wherein the polymerase enzyme complexes are bound to the chip through a modified biotin-binding protein that comprises one or more covalently attached sulfonate moieties;

sequencing reagents in contact with the surface comprising reagents for carrying out nucleic acid synthesis including one or more labeled nucleotide analogs;

an illumination system for illuminating the polymerase enzyme complexes;

an optical detection system for detecting fluorescence from the labeled nucleotide analogs while they are interacting with the polymerase enzyme complexes; and

a computer for analyzing the signals detected by the detection system to determine the sequential addition of nucleotides to a nucleic acid strand complementary to a strand of the template nucleic acid.