Conjugates of biomolecules to nanoparticles

Patent metadata

Field	Value
Publication number	US-11015220-B2
Application number	US-201816123166-A
Country	US
Kind code	B2
Filing date	Sep 6, 2018
Priority date	Mar 27, 2009
Publication date	May 25, 2021
Grant date	May 25, 2021

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.

First claim

What is claimed is:

1. A composition, comprising a population of polymerase-nanoparticle conjugates including a polymerase linked to a nanoparticle, wherein the population of polymerase-nanoparticle conjugates has an average of about 0.5 to 1.5 polymerases per nanoparticle and wherein the conjugate has polymerase activity that is at least 70%-relative to the polymerase activity of the unconjugated polymerase;
wherein the nanoparticle is capable of generating a detectable signal.

2. The composition of claim 1 , wherein the polymerase activity of the conjugate is at least 80%, 90%, 95%, 97% or 99% relative to the polymerase activity of the unconjugated polymerase.

3. The composition of claim 1 , wherein there is a 1:1 correspondence between the polymerase and the nanoparticle.

4. The composition of claim 1 , further comprising an accessory compound selected from among uracil DNA glycosylase, uracil DNA glycosylase inhibitor maltose binding protein, albumin, avidin, chloramphenicol acetyltransferase, horseradish peroxidase, glutathione S-transferase and mucin.

5. The composition of claim 3 , further comprising an accessory compound selected from among uracil DNA glycosylase, uracil DNA glycosylase inhibitor, maltose binding protein, albumin, avidin, chloramphenicol acetyltransferase, horseradish peroxidase, glutathione S-transferase and mucin.

6. The composition of claim 4 , wherein the accessory compound is attached to the nanoparticle.

7. The composition of claim 6 , wherein the accessory compound is a protein that does not react with the polymerase or nucleotides.

8. The composition of claim 4 , wherein the accessory compound further comprises a His tag.

9. The composition of claim 1 , wherein the nanoparticle is positioned relative to the polymerase to perform an energy transfer reaction with a labeled nucleotide bound to the nucleotide binding site of the polymerase.

10. The composition of claim 1 , wherein the nanoparticle comprises a nanocrystal comprising a semiconductor.

11. A composition, comprising a population of biomolecule-nanoparticle conjugates that include a polymerase linked to a nanoparticle, wherein an at least about 20% of the polymerase-nanoparticle conjugates in the population have an average of about 0.5-1.5 polymerase biomolecules per conjugate;
wherein the nanoparticle is capable of generating a detectable signal.

12. The composition of claim 11 , comprising: a polymerase including a His-tag-linked to the nanoparticle via a His-tag mediated attachment.

13. The composition of claim 11 , wherein at least about 40% of the biomolecule-nanoparticle conjugates in the population are monodisperse conjugates.

14. The composition of claim 11 , wherein the biomolecule is a polymerase.

15. The composition of claim 11 , further comprising an accessory compound selected from among uracil DNA glycosylase, uracil DNA glycosylase, maltose binding protein, albumin, avidin, chloramphenicol acetyltransferase, horseradish peroxidase, glutathione S-transferase and mucin.

16. The composition of claim 15 , wherein the accessory compound is a protein that does not react with a polymerase or nucleotides.

17. The composition of claim 11 , wherein the nanoparticle comprises a nanocrystal comprising a semiconductor.

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

19. 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.

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

21. The composition of claim 20 , 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.

22. 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.

23. 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.

24. 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.

Assignees

• Life Technologies Corp

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

Classifications

• C07H19/20 Primary

  with the saccharide radical esterified by phosphoric or polyphosphoric acids · CPC title

• C12N9/1252

  DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase · CPC title

• C12Y207/07

  Nucleotidyltransferases (2.7.7) · CPC title

• G01N21/6428

  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 title

• C12Y207/07007

  DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase · CPC title