Modified biotin-binding proteins for immobilization

Patent metadata

Field	Value
Publication number	US-11781177-B2
Application number	US-202016841450-A
Country	US
Kind code	B2
Filing date	Apr 6, 2020
Priority date	Dec 22, 2017
Publication date	Oct 10, 2023
Grant date	Oct 10, 2023

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.

First claim

What is claimed is:

1. A system for sequencing nucleic acids, the system comprising
a chip comprising a plurality of polymerase enzyme complexes bound thereto, wherein each of the polymerase enzyme complexes is individually optically resolvable, wherein each of the polymerase enzyme complexes comprises 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 a surface of the chip through a modified biotin-binding protein that comprises three or more covalently attached sulfonate moieties;
sequencing reagents in contact with the surface of the chip comprising reagents for carrying out nucleic acid synthesis including one or more fluorescently labeled nucleotide analogs;
an illumination system for illuminating the polymerase enzyme complexes;
an optical detection system for detecting fluorescent signals from the fluorescently labeled nucleotide analogs while they are interacting with the polymerase enzyme complexes; and
a computer for analyzing the fluorescent signals detected by the optical detection system to determine the sequential addition of the one or more fluorescently labeled nucleotide analogs to a nucleic acid strand complementary to a strand of the template nucleic acid.

2. The system of claim 1 , wherein the chip comprises a plurality of nanoscale reaction regions that comprises the polymerase enzyme complexes.

3. The system of claim 1 , wherein the chip comprises a plurality of nanoscale wells that comprises the polymerase enzyme complexes.

4. The system of claim 1 , wherein the chip comprises a plurality of nanoscale wells, wherein each of the plurality of nanoscale wells includes a single active polymerase enzyme complex from the plurality of polymerase enzyme complexes immobilized at its base.

5. The system of claim 1 , wherein the modified biotin-binding protein is a tetrameric biotin-binding protein.

6. The system of claim 1 , wherein the modified biotin-binding protein is traptavidin.

7. The system of claim 1 , wherein the modified biotin-binding protein is a tetravalent biotin-binding protein.

8. The system of claim 1 , wherein the modified biotin-binding protein is streptavidin.

9. The system of claim 1 , wherein the three or more covalently attached sulfonate moieties are 12 or more covalently attached sulfonate moieties.

10. The system of claim 1 , wherein the three or more covalently attached sulfonate moieties are 24 or more covalently attached sulfonate moieties.

11. The system of claim 1 , wherein the three or more covalently attached sulfonate moieties are 30 or more covalently attached sulfonate moieties.

12. The system of claim 1 , wherein the three or more covalently attached sulfonate moieties are 45 or more covalently attached sulfonate moieties.

13. The system of claim 1 , wherein the three or more covalently attached sulfonate moieties comprise one or more covalently attached 3,4,5-tris(3-sulfopropoxy)benzoyl moieties.

14. The system of claim 1 , wherein the three or more covalently attached sulfonate moieties comprise four or more covalently attached 3,4,5-tris(3-sulfopropoxy)benzoyl moieties.

15. The system of claim 1 , wherein the three or more covalently attached sulfonate moieties comprise 10 or more covalently attached 3,4,5-tris(3-sulfopropoxy)benzoyl moieties.

16. The system of claim 1 , wherein the three or more covalently attached sulfonate moieties comprise 15 or more covalently attached 3,4,5-tris(3-sulfopropoxy)benzoyl moieties.

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

18. The system of claim 17 , wherein the modified biotin-binding protein is streptavidin.

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

20. The system of claim 19 , wherein the modified biotin-binding protein is streptavidin.

21. The system of claim 1 , wherein the three or more covalently attached sulfonate moieties comprise one or more covalently attached 3,5-disulfobenzoyl or 2-sulfobenzoyl moieties.

22. The system of claim 1 , wherein the modified biotin-binding protein comprises one or more amino acid substitutions that decrease its calculated net charge at pH 7.4 relative to a biotin-binding protein that is used to make the modified biotin-binding protein.

Assignees

• Pacific Biosciences California Inc

Inventors

• Kamtekar Satwik
• Sebo Lubomir
• Chern Leewin
• Linsky Thomas
• Hanes Jeremiah
• Miller Erik
• Yang Ying
• Yue Stephen

Classifications

• C12N9/22 Primary

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title

• C12Q1/6848 Primary

  characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction · CPC title

• C07K14/36

  from Actinomyces; from Streptomyces (G) · CPC title

• C12Q2535/00

  Reactions characterised by the assay type for determining the identity of a nucleotide base or a sequence of oligonucleotides · CPC title

• C12Q2537/143

  Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis · CPC title