Conjugates of biomolecules to nanoparticles

• US 9,365,838 B2
• Filed: 11/22/2013
• Issued: 06/14/2016
• 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: Lincecum, Tommie Lloyd, Mauro, John, Nikiforov, Theo, Mazur, Daniel, Peng, Xinzhan, Belosludtsev, Yuri, Reese, Howard, Gremyachinskiy, Dmitriy, Rozhkov, Roman, Beechem, Joseph, Tulsky, Eric, Naasani, Imad, Haley, Kari, Treadway, Joseph
• Primary Examiner(s): DESAI, ANAND U
• Application Number:
  US14/087,307
• Publication Number:
  US 20140308730A1
• Time in Patent Office: 935 Days
• Field of Search: None
• US Class Current: 1/1
• 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 composition, comprising a polymerase-nanoparticle conjugate including a polymerase linked to a nanoparticle, wherein the conjugate has polymerase activity, and wherein the polymerase-nanoparticle conjugate includes a template nucleic acid molecule, a polymerization initiation site, and lacks a helicase.

2. The composition of claim 1, wherein the polymerase activity of the conjugate is at least 1% relative to the polymerase activity of the unconjugated polymerase.

3. The composition of claim 2, wherein the polymerase activity is at least 70% relative to the polymerase activity of the unconjugated polymerase.

4. The composition of claim 1, wherein the polymerase comprises a His tag.

5. The composition of claim 1, wherein the polymerase comprises one member of a binding pair and the nanoparticle comprises a complementary member of the binding pair.

6. The composition of claim 1, wherein the polymerase is a DNA polymerase.

7. The composition of claim 6, wherein the DNA polymerase is at least 95% identical to a DNA polymerase selected from the group consisting of:

Phi-29 DNA polymerase, B103 DNA polymerase, the Klenow fragment of E. coli DNA polymerase and HIV reverse transcriptase.

8. The composition of claim 1, wherein the polymerase link is selected from one of a group consisting of:

covalent bonding, affinity bonding and electrostatic bonding.

9. The composition of claim 1, wherein the nanoparticle has a surface coating including a ligand selected from the group consisting of:

a DHLA-based ligand, a polycyclic acid-based ligand, a bipeptide ligand and a tridentate thiol-based ligand.

10. The composition of claim 1, wherein the nanoparticle includes a DHLA-based ligand coating.

11. The composition of claim 1, wherein the nanoparticle includes a polycyclic acid-based ligand coating.

12. The composition of claim 1, wherein the nanoparticle includes a tripodal thiol-based ligand coating.

13. The composition of claim 1, wherein the nanoparticle includes a dipeptide-based coating.

14. The composition of claim 1, wherein the nanoparticle is positioned relative to the polymerase to perform an energy transfer reaction.

15. The composition of claim 1, wherein the nanoparticle is positioned to undergo FRET with a labeled nucleotide bound to the nucleotide binding site of the polymerase.

16. The composition of claim 15, wherein labeled nucleotide further comprises an Alexa Fluor dye linked to the terminal phosphate of the nucleotide, and the nanoparticle undergoes FRET with the Alexa Fluor dye with a FRET efficiency of at least 20%.

17. The composition of claim 1, where the polymerase includes an N-terminal cysteine, and where N-terminal cysteine is linked to the nanoparticle through a covalent bond.

18. The composition of claim 17, wherein the covalent bond is formed between a thioester surface ligand of the nanoparticle and a thiol group of the N-terminal cysteine.

19. The composition of claim 17, wherein the covalent bond is formed between an aldehyde surface ligand of the nanoparticle and a thiol group of the N-terminal cysteine.

20. The composition of claim 1, further comprising a template nucleic acid molecule and a nucleotide.