Polymerase enzyme substrates with protein shield

• US 9,062,091 B2
• Filed: 02/14/2013
• Issued: 06/23/2015
• Est. Priority Date: 02/15/2012
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Bjornson, Keith, Hanes, Jeremiah, Miller, Erik, Kamtekar, Satwik, Sebo, Lubomir, Brogley, Louis
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US13/767,619
• Publication Number:
  US 20130316912A1
• Time in Patent Office: 859 Days
• Field of Search: 536/26.6, 435/6.1, 435/91.1, 422/430, 422/68.1
• US Class Current: 1/1
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 19/16 Purine radicals
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C07K 14/36 from Actinomyces; from Streptomyces (G)
• CPC Class Codes: C09B 23/083 five >CH- groups
• CPC Class Codes: C09B 69/105 containing a methine or polymethine dye
• CPC Class Codes: C12N 9/10 Transferases (2.) ribonucleases C12N9/22
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2523/313 Irradiation, e.g. UV irradiation
• CPC Class Codes: C12Q 2535/113 Cycle sequencing
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: G01N 33/583 with non-fluorescent dye label

Abstract

Compositions and methods are provided for nucleotide analogs comprising protein shields for improving enzyme photostability in single molecule real time sequencing. Nucleotide analogs of the invention have a protein shield between the dye moieties and nucleotide moieties of the analog. The protein prevents the direct interaction of the dye moiety with the enzyme carrying out nucleotide synthesis preventing photodamage to the enzyme. The nucleotide analogs of the invention can have multiple dyes and multiple nucleotide moieties.

Claims

1. A labeled nucleotide analog comprising:

an avidin protein having four subunits, each subunit comprising one biotin binding site;

one or two nucleotide components bound to the avidin protein, each comprising one or more phospholinked nucleotide moieties; and

one or two dye components, bound to the avidin protein, each comprising one or more dye moieties;

wherein each component is bound to the avidin protein through a biotin moiety attached to a binding site on the avidin protein; and

wherein at least one of the nucleotide or dye components comprise a bis-biotin moiety bound to two of the biotin binding sites on the avidin protein.

2. The labeled nucleotide analog of claim 1 wherein the nucleotide analog has one nucleotide component that comprises a bis-biotin moiety, and the labeled nucleotide analog has two dye components each bound to the avidin through a single biotin moiety.

3. The labeled nucleotide analog of claim 1 wherein the nucleotide analog has one dye component that comprises a bis-biotin moiety, and the labeled nucleotide analog has two nucleotide components each bound to the avidin through a single biotin moiety.

4. The labeled nucleotide analog of claim 1 wherein the labeled nucleotide analog has one dye component and one nucleotide component and each of the dye component and the nucleotide component comprises a bis-biotin moiety.

5. The labeled nucleotide analog of claim 1 wherein the number of dye moieties is between 1 and 18 and the number of nucleotide moieties is between 1 and 18.

6. The labeled nucleotide analog of claim 1 wherein the number of dye moieties is 1, 2, or 3 and the number of nucleotide moieties is 6, 7, or 8.

7. The labeled nucleotide analog of claim 1 wherein the number of bonds between biotins on the bis-biotin moiety is between 15 and 50.

8. The labeled nucleotide analog of claim 1 wherein the avidin protein comprises either streptavidin or tamavidin.

9. The labeled nucleotide analog of claim 1 wherein the dye moieties comprise fluorescent labels.

10. A reaction mixture for sequencing a nucleic acid template comprising:

a 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 complex is immobilized on a surface; and

sequencing reagents in contact with the surface comprising reagents for carrying out nucleic acid synthesis including 2 or more labeled nucleotide analogs of claim 1.

11. The reaction mixture of claim 10 wherein a labeled nucleotide analog has one dye component and one nucleotide component and each of the dye component and the nucleotide component comprises a bis-biotin moiety.

12. A method for sequencing a nucleic acid template comprising:

providing a 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 complex is immobilized on a surface;

adding sequencing reagents in contact with the surface comprising reagents for carrying out nucleic acid synthesis including 2 or more labeled nucleotide analogs of claim 1; and

determining the sequential addition of nucleotides to a nucleic acid strand complementary to a strand of the template nucleic acid by observing the interaction of the labeled nucleotide analogs with the polymerase enzyme complex.

13. The method of claim 12 wherein the nucleotide component comprises a bis-biotin moiety, and a labeled nucleotide analog has two dye components each bound to the avidin through a single biotin moiety.

14. The method of claim 12 wherein the dye component comprises a bis-biotin moiety, and a labeled nucleotide analog has two nucleotide components each bound to the avidin through a single biotin moiety.

15. The method of claim 12 wherein a labeled nucleotide analog has one dye component and one nucleotide component and each of the dye component and the nucleotide component comprises a bis-biotin moiety.

16. The method of claim 12 wherein the number of dye moieties is between 1 and 18 and the number of nucleotide moieties is between 1 and 18 in each nucleotide analog.

17. The method of claim 12 wherein the number of dye moieties is 1, 2, or 3 and the number of nucleotide moieties is 6, 7, or 8 in each nucleotide analog.

18. The method of claim 12 wherein the number of bonds between biotins on a bis-biotin moiety is between 15 and 50.

19. The method of claim 12 wherein the avidin protein in an analog comprises either streptavidin or tamavidin.

20. The method of claim 12 wherein the dye moieties comprise fluorescent labels.

21. A system for sequencing nucleic acids 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,

sequencing reagents in contact with the surface comprising reagents for carrying out nucleic acid synthesis including 2 or more labeled nucleotide analogs of claim 1; and

an illumination system for illuminating the polymerase enzyme complexes; and

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.