Labeled enzyme compositions, methods and systems

• US 9,932,573 B2
• Filed: 01/09/2017
• Issued: 04/03/2018
• Est. Priority Date: 03/27/2009
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Nikiforov, Theo
• Primary Examiner(s): Hutson, Richard G
• Application Number:
  US15/401,757
• Publication Number:
  US 20170191051A1
• Time in Patent Office: 449 Days
• Field of Search: None
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C12N 9/1241 Nucleotidyltransferases (2.7.7)
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12N 9/96 Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Y 207/07 Nucleotidyltransferases (2.7.7)
• CPC Class Codes: C12Y 207/07007 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: G01N 2021/6432 Quenching
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 33/582 with fluorescent label

Abstract

Disclosed herein are conjugates comprising a biomolecule linked to a label that have biological activity and are useful in a wide variety of biological applications. For example, provided herein are labeled polymerase conjugates including a polymerase linked to one or more labels, wherein the conjugate has polymerase activity. Such conjugates can exhibit enhanced biological activity and/or superior detectability as compared to conventional labeled polymerases. Also disclosed herein are improved methods for preparing such conjugates, and methods and systems for using such conjugates in biological applications such as nucleotide incorporation, primer extension and single molecule sequencing.

Claims

1. A method for creating a labeled polymerase conjugate, comprising:

linking a polymerase to at least three optically-detectable dye labels to form a labeled polymerase conjugate having polymerase activity and the at least three dye labels linked to the polymerase,

wherein the polymerase includes a biotin ligase modification site, and the linking comprises linking a biotin moiety to the polymerase to produce a biotinylated polymerase.

2. The method of claim 1, wherein the linking comprises linking the polymerase to four, five, six, or seven optically-detectable dye labels.

3. The method of claim 1, wherein the linking comprises linking the at least three optically-detectable dye labels to an attachment site on the polymerase.

4. The method of claim 1, wherein the linking comprises linking the at least three optically-detectable dye labels to independent attachment sites on the polymerase.

5. The method of claim 1, wherein the linking further comprises contacting the biotinylated polymerase with an avidin moiety linked to the at least three optically-detectable dye labels under conditions where the avidin moiety binds to the biotin moiety.

10. The method of claim 1, wherein the labeled polymerase conjugate has activity that is at least about 80% relative to the polymerase activity of the unconjugated polymerase.

11. The method of claim 1, wherein the labeled polymerase conjugate emits upon continuous excitation a total photon count of at least 10² photons before irreversibly photobleaching.

12. The method of claim 11, wherein the labeled polymerase conjugate emits a total photon count of at least 10⁶ photons as measured using a test detection system.

13. The method of claim 12, wherein the labeled polymerase conjugate emits a total photon count of at least 10⁸ photons as measured using a test detection system.

14. The method of claim 1, wherein at least one of the linked optically-detectable dye labels is positioned to undergo FRET with a labeled nucleotide bound to the nucleotide binding site of the labeled polymerase conjugate.

15. The method of claim 14, wherein at least three of the linked optically-detectable dye labels are positioned to undergo FRET with a labeled nucleotide bound to the nucleotide binding site of the labeled polymerase conjugate.

6. A method for creating a labeled polymerase conjugate, comprising:

linking a polymerase to at least three optically-detectable dye labels to form a labeled polymerase conjugate having polymerase activity and the at least three dye labels linked to the polymerase, wherein the polymerase includes one or more biotin acceptor peptide sequence.

7. The method of claim 6, wherein at least one of the optically-detectable dye labels is linked to one biotin acceptor peptide sequence.

8. The method of claim 7, wherein the at least three optically-detectable dye labels are linked to one biotin acceptor peptide sequence of the polymerase.

9. The method of claim 6, wherein the at least three optically-detectable dye labels are linked to a plurality of biotin acceptor peptide sequences of the polymerase.