Conjugates of biomolecules to nanoparticles

• US 8,603,792 B2
• Filed: 03/26/2010
• Issued: 12/10/2013
• 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, Belosludtsev, Yuri, Reese, Howard, Gremyachinskiy, Dmitriy, Rozhkov, Roman, Mauro, John M., Beechem, Joseph, Tulsky, Eric, Naasani, Imad, Haley, Kari, Treadway, Joseph A., Lincecum, Tommie Lloyd Jr.
• Primary Examiner(s): DESAI, ANAND U
• Application Number:
  US12/748,355
• Publication Number:
  US 20110003343A1
• Time in Patent Office: 1,355 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 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 performing a primer extension reaction, comprising:

contacting a polymerase nanoparticle conjugate with a nucleic acid molecule and a nucleotide under conditions where the polymerase of the conjugate extends the nucleic acid molecule by a nucleotide, the polymerase nanoparticle conjugate including a polymerase linked to a nanoparticle and the conjugate having polymerase activity.

2. The method of claim 1, wherein the nucleotide further comprises a label linked to a terminal phosphate group of the nucleotide.

3. The method of claim 2, further comprising detecting a signal resulting from FRET between the nanoparticle and the label of the nucleotide.

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

5. The method of claim 4, wherein the polymerase activity is at least 70% relative to the polymerase activity of the unconjugated polymerase.

6. The method of claim 1, wherein the polymerase comprises a His tag.

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

8. The method of claim 1, wherein the polymerase is a DNA polymerase.

9. The method of claim 8, 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.

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

covalent bonding, affinity bonding and electrostatic bonding.

11. The method 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.

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

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

14. The method of claim 1, wherein the nanoparticle includes a tridentate thiol-based ligand coating.

15. The method of claim 1, wherein the nanoparticle includes a bipeptide-based coating.

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

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

18. The method of claim 17, 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 about 20%.

19. The method 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.

20. The method of claim 19, wherein the covalent bond is formed between a thioester surface ligand of the nanoparticle and a thiol group of the N-terminal cysteine.

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

22. The method of claim 1, wherein the polymerase activity of the conjugate is about 1% relative to the polymerase activity of the unconjugated polymerase.

23. The method of claim 4, wherein the polymerase activity is about 70% relative to the polymerase activity of the unconjugated polymerase.