Multivalent binding composition for nucleic acid analysis

Patent metadata

Field	Value
Publication number	US-12313627-B2
Application number	US-202418431748-A
Country	US
Kind code	B2
Filing date	Feb 2, 2024
Priority date	May 1, 2019
Publication date	May 27, 2025
Grant date	May 27, 2025

Abstract

Multivalent binding compositions including a particle-nucleotide conjugate having a plurality of copies of a nucleotide attached to the particle are described. The multivalent binding compositions allow one to localize detectable signals to active regions of biochemical interaction, e.g., sites of protein-protein interaction, protein-nucleic acid interaction, nucleic acid hybridization, or enzymatic reaction, and can be used to identify sites of base incorporation in elongating nucleic acid chains during polymerase reactions and to provide improved base discrimination for sequencing and array based applications.

First claim

What is claimed:

1. A method for nucleic acid hybridization, the method comprising:
(a) providing a surface comprising a hydrophilic polymer coating layer coupled thereto, wherein the hydrophilic polymer coating layer comprises a first nucleic acid molecule coupled thereto, and wherein the hydrophilic polymer coating layer has a water contact angle of less than 45 degrees; and
(b) bringing the first nucleic acid molecule coupled to the hydrophilic polymer coating layer into contact with a hybridizing composition comprising (i) a second nucleic acid molecule at a concentration of one nanomolar or less and (ii) at least one organic solvent that is polar and aprotic, under conditions sufficient for the second nucleic acid molecule to hybridize to the first nucleic acid molecule coupled to the hydrophilic polymer coating layer in 30 minutes or less, wherein the method uses a reduced concentration of the second nucleic acid molecule and results in a shortened hybridization time as compared to a method that is not performed using the hybridizing composition and the surface.

2. The method of claim 1 , wherein (b) is performed at a temperature that is from about 30 degrees Celsius to 70 degrees Celsius.

3. The method of claim 1 , further comprising performing a nucleotide binding reaction on the surface between the first nucleic acid molecule and the second nucleic acid molecule.

4. The method of claim 3 , wherein the pH buffer comprises 2-(N-morpholino)ethanesulfonic acid, acetonitrile, 3-(N-morpholino)propanesulfonic acid, methanol, or a combination thereof.

5. The method of claim 1 , wherein the second nucleic acid molecule is present in the hybridizing composition at a concentration of 0.50 nanomolar or less.

6. The method of claim 1 , wherein the second nucleic acid molecule is present in the hybridizing composition at a concentration of 250 picomolar or less.

7. The method of claim 1 , wherein the second nucleic acid molecule is present in the hybridizing composition at a concentration of 100 picomolar or less.

8. The method of claim 1 , wherein bringing the first nucleic acid molecule coupled to the hydrophilic polymer coating layer into contact with the hybridizing composition is performed for a time period of less than 30 minutes.

9. The method of claim 1 , further comprising hybridizing the second nucleic acid molecule to the first nucleic molecule coupled to the hydrophilic polymer coating layer at a hybridization efficiency that is increased as compared to a comparable hybridization reaction performed for 120 minutes at 90 degrees Celsius for 5 minutes followed by cooling for 120 minutes to reach a final temperature of 37 degrees Celsius in a buffer comprising saline-sodium citrate.

10. The method of claim 1 , further comprising hybridizing the second nucleic acid molecule to the first nucleic acid molecule with a hybridization stringency of at least 80%.

11. The method of claim 1 , wherein the hydrophilic polymer coating layer exhibits a level of non-specific Cyanine 3 dye absorption of less than about 0.25 molecules per square micrometer.

12. The method of claim 1 , wherein the hybridizing composition further comprises a pH buffer.

13. The method of claim 1 , wherein the at least one organic solvent has a dielectric constant of no greater than about 115 as measured at 68 degrees Fahrenheit.

14. The method of claim 1 , wherein the at least one organic solvent comprises at least one functional group selected from the group consisting of nitrile, lactone, sulfone, sulfite, and carbonate.

15. The method of claim 1 , wherein the hybridizing composition further comprises formamide.

16. The method of claim 1 , wherein the at least one organic solvent is miscible with water.

17. The method of claim 1 , wherein a concentration of the at least one organic solvent in the hybridizing composition is at least about 5% by volume based on a total volume of the hybridizing composition.

18. The method of claim 1 , wherein a concentration of the at least one organic solvent in the hybridizing composition is at least about 95% by volume based on a total volume of the hybridizing composition.

19. The method of claim 1 , wherein the first nucleic acid molecule is coupled to the hydrophilic polymer coating layer through covalent bonding.

20. The method of claim 1 , wherein the hybridizing composition further comprises a crowding agent.

21. The method of claim 20 , wherein the crowding agent is selected from the group consisting of polyethylene glycol, dextran, hydroxypropyl methyl cellulose, hydroxyethyl methyl cellulose, hydroxybutyl methyl cellulose, hydroxypropyl cellulose, methyl cellulose, and hydroxyl methyl cellulose, and any combination thereof.

Assignees

• Element Biosciences Inc

Inventors

• Arslan Sinan
• He Molly
• Previte Michael

Classifications

• G01N33/5308 Primary

  for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites · CPC title

• G01N33/582

  with fluorescent label · CPC title

• C12Q1/6874

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title

• C12Q1/6869 Primary

  Methods for sequencing · CPC title