Labeled enzyme compositions, methods and systems

• US 8,741,618 B2
• Filed: 08/24/2012
• Issued: 06/03/2014
• 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:
  US13/594,532
• Publication Number:
  US 20130040363A1
• Time in Patent Office: 648 Days
• Field of Search: None
• US Class Current: 435/194
• 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 labeled polymerase conjugate, comprising:

a polymerase linked to at least three optically detectable dye labels, wherein the at least three optically detectable dye labels are linked to one or more biotin acceptor peptide sequences of the polymerase, and wherein the conjugate has polymerase activity and emits upon continuous excitation a total photon count of at least 10⁶ photons before irreversibly photobleaching.

2. The conjugate of claim 1, wherein the polymerase is linked to three, four, five, six or seven optically detectable dye labels.

3. The conjugate of claim 1, wherein the conjugate emits a total photon count of at least 10⁸ photons as measured using a test detection system.

4. The conjugate of claim 1, wherein the at least three optically detectable dye labels are linked to one biotin acceptor peptide sequence of the polymerase.

5. The conjugate of claim 1, wherein the at least three optically detectable dye labels are linked to a plurality of biotin acceptor peptide sequences of the polymerase.

6. The conjugate of claim 1, wherein the biotin acceptor peptide sequence comprises the amino acid sequence of SEQ ID NO:

10.

7. The conjugate of claim 1, wherein the polymerase activity is at least about 80% relative to the polymerase activity of the unconjugated polymerase.

8. The conjugate of claim 1, wherein the at least three optically detectable dye labels linked to the polymerase undergo energy transfer with a labeled nucleotide bound to the nucleotide binding site of the polymerase.

9. The conjugate of claim 8, wherein the labeled nucleotide comprises a label linked to the terminal phosphate of the nucleotide.

10. The conjugate of claim 8, wherein the at least three optically detectable dye labels linked to the polymerase undergo Forster Resonance Energy Transfer (FRET) with the labeled nucleotide bound to the nucleotide binding site of the polymerase.