Conjugates of biomolecules to nanoparticles

• US 10,093,972 B2
• Filed: 05/27/2016
• Issued: 10/09/2018
• 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, Mazur, Daniel, Peng, Xinzhan, Lincecum, Tommie Lloyd, Belosludtsev, Yuri, Reese, Howard, Gremyachinskiy, Dmitriy, Rozhkov, Roman, Mauro, John, Beechem, Joseph, Tulsky, Eric, Naasani, Imad, Haley, Kari, Treadway, Joseph
• Primary Examiner(s): Desai, Anand U
• Application Number:
  US15/166,372
• Publication Number:
  US 20160265044A1
• Time in Patent Office: 865 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 polymerase-nanoparticle conjugates including a polymerase linked to a nanoparticle, wherein the conjugate has polymerase activity. Such conjugates can exhibit reduced aggregation and improved stochiometries wherein the average biomolecule:nanoparticle ratio approaches or equals 1:1. 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 preparing a population of polymerase-nanoparticle conjugates which comprise a polymerase linked to a nanoparticle, where the polymerase includes a His-tag at the N-terminal end, and where the polymerase is linked to the nanoparticle via a His-tag mediated attachment, thereby forming a polymerase-nanoparticle conjugate, and where the conjugate has polymerase activity, wherein the polymerase-nanoparticle conjugate is prepared by:

a) producing a polymerase including a His-tag, and

b) contacting the polymerase with a nanoparticle and a quantity of an accessory compound under conditions where the polymerase becomes linked to the nanoparticle via a His-tag mediated attachment, thereby forming a a population of polymerase-nanoparticle conjugates with an average of about 0.5 to 1.5 polymerases per nanoparticle, wherein the accessory compound is selected from a group consisting of horseradish peroxidase, mucin, albumin, avidin, chloramphenicol acetyl-transferase, maltose binding protein and uracil DNA glycosylase or a combination thereof.

2. The method of claim 1 , wherein the resulting population of polymerase-nanoparticle conjugates comprises at least about 20% of the conjugates including an average of 1 polymerase per nanoparticle, relative to total number of nanoparticles in the population.

3. The method of claim 1, wherein the nanoparticles comprise CdSe-ZnS core-shell nanoparticles.

4. The method of claim 1, wherein the DNA polymerase comprises Phi29 DNA polymerase.

5. The method of claim 1, wherein the plurality of nanoparticles is contacted in step (b) with histidine tagged uracil DNA glycosylase (UDG) and uracil DNA glycosylase inhibitor (ugi) to generate histidine tagged UDG-ugi conjugates.

6. The method of claim 5, wherein the plurality of nanoparticles is contacted in step (b) with the histidine-tagged UDG-ugi conjugates in a 1:

18 molar ratio of nanoparticles to histidine-tagged UDG-ugi conjugates.

7. The method of claim 5, wherein the plurality of nanoparticles is contacted in step (b) with the histidine-tagged UDG-ugi conjugates in a 1:

16 molar ratio nanoparticles to histidine-tagged UDG-ugi conjugates.

8. The method of claim 5, wherein the plurality of histidine-tagged UDG-ugi nanoparticle conjugates in step (c) is contacted with the histidine-tagged DNA polymerases in a 1:

4 molar ratio of histidine-tagged UDG-ugi nanoparticle conjugates to histidine-tagged DNA polymerases.

9. The method of claim 5, wherein the plurality of histidine-tagged UDG-ugi nanoparticle conjugates in step (c) is contacted with the histidine-tagged DNA polymerases in a 1:

2 molar ratio of histidine-tagged UDG-ugi nanoparticle conjugates to histidine-tagged DNA polymerases.

10. The method of claim 5, wherein the plurality of histidine-tagged UDG-ugi nanoparticle conjugates in step (c) is contacted with the histidine-tagged DNA polymerases in a 1:

1 molar ratio of histidine-tagged UDG-ugi nanoparticle conjugates to histidine-tagged DNA polymerases.