Modified biotin-binding proteins for immobilization

Patent metadata

Field	Value
Publication number	US-2020354783-A1
Application number	US-202016841450-A
Country	US
Kind code	A1
Filing date	Apr 6, 2020
Priority date	Dec 22, 2017
Publication date	Nov 12, 2020
Grant date	—

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

1 . (canceled)

2 . The system of claim 152 , wherein the biotin-binding protein is a tetravalent biotin-binding protein.

3 . The system of claim 152 , wherein the biotin-binding protein is streptavidin.

4 . (canceled)

5 . The system of claim 152 , wherein the biotin-binding protein comprises 12 or more covalently attached sulfonate moieties.

6 . The system of claim 152 , wherein the biotin-binding protein comprises 24 or more covalently attached sulfonate moieties.

7 . The system of claim 152 , wherein the biotin-binding protein comprises 30 or more covalently attached sulfonate moieties.

8 . The system of claim 152 , wherein the biotin-binding protein comprises 45 or more covalently attached sulfonate moieties.

9 . (canceled)

10 . The system of claim 152 , wherein the biotin-binding protein comprises one or more covalently attached 3,4,5-tris(3-sulfopropoxy)benzoyl moieties.

11 . The system of claim 152 , wherein the biotin-binding protein comprises four or more covalently attached 3,4,5-tris(3-sulfopropoxy)benzoyl moieties.

12 . The system of claim 152 , wherein the biotin-binding protein comprises 10 or more covalently attached 3,4,5-tris(3-sulfopropoxy)benzoyl moieties.

13 . The system of claim 152 , wherein the biotin-binding protein comprises 15 or more covalently attached 3,4,5-tris(3-sulfopropoxy)benzoyl moieties.

14 . (canceled)

15 . The system of claim 152 , wherein the biotin-binding protein is a tetravalent biotin-binding protein comprising 45 or more covalently attached sulfonate moieties.

16 . The system of claim 15 , wherein the biotin-binding protein is streptavidin.

17 . The system of claim 152 , 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.

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

19 . The system of claim 152 , wherein the biotin-binding protein comprises one or more covalently attached 3,5-disulfobenzoyl or 2-sulfobenzoyl moieties.

20 . (canceled)

21 . The system of claim 152 , 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.

22 - 151 . (canceled)

152 . 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 three 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.

153 . The system of claim 152 , wherein the chip comprises a plurality of nanoscale reaction regions that comprise the polymerase enzyme complexes.

154 . The system of claim 152 , wherein the chip comprises a plurality of nanoscale wells that comprise the polymerase enzyme complexes.

155 . The system of claim 154 , wherein a plurality of the nanoscale wells include a single active polymerase enzyme complex immobilized at the base of the well.

156 . The system of claim 152 , wherein the biotin-binding protein is a tetrameric biotin-binding protein.

157 . The system of claim 152 , wherein the biotin-binding protein is traptavidin.

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

• C12Q2537/143

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

• C07K14/36

  from Actinomyces; from Streptomyces (G) · CPC title

• C12Q1/6848 Primary

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