Methods and compositions for coupling nucleic acid to a functionalized support

Patent metadata

Field	Value
Publication number	US-11555183-B2
Application number	US-201816645896-A
Country	US
Kind code	B2
Filing date	Aug 30, 2018
Priority date	Sep 11, 2017
Publication date	Jan 17, 2023
Grant date	Jan 17, 2023

Abstract

This invention relates to methods and compositions for coupling nucleic acid to a functionalized surface or support. In particular, the present invention provides an improved process for coupling aminated nucleic acid to a support functionalized with carboxylic acid groups, wherein the coupling reaction is conducted in the presence of an organic solvent. The invention further relates to compositions and kits for performing the coupling reaction and uses of nucleic acid-loaded supports for various applications.

First claim

The invention claimed is:

1. A method for coupling a nucleic acid to a support comprising:
(a) providing a support comprising thereon carboxylic acid groups,
(b) providing a nucleic acid, wherein the nucleic acid comprises at least one primary amine function,
(c) activating the support by contacting the carboxylic acid groups thereon with a carbodiimide or a derivative thereof, to form a reactive anhydride,
(d) reacting the reactive anhydride with the at least one primary amine function of the nucleic acid to form a covalent amide bond, thereby coupling said nucleic acid to the support,
wherein steps (c) and (d) are conducted in the presence of an organic solvent.

2. The method of claim 1 , wherein steps (c) and (d) are conducted in the presence of a salt.

3. The method of claim 1 , wherein the organic solvent is selected from the group consisting of dimethyl sulfoxide, dimethylformamide, and diethyl sulfoxide or a mixture thereof.

4. The method of claim 1 , wherein the organic solvent is present at step (c) and/or step (d) at a concentration of at least about 50% and less than 95% per total volume.

5. The method of claim 1 , wherein step (c) and/or step (d) are conducted in the presence of water or an aqueous buffer, wherein the concentration of water or aqueous buffer is less than 20%, preferably less than 10% per total volume.

6. The method of claim 1 , wherein the carbodiimide is EDC.

7. The method of claim 1 , wherein the carbodiimide or derivative thereof is dissolved in aqueous buffer prior to use in step (c).

8. The method of claim 7 , wherein the aqueous buffer is MES or imidazole buffer.

9. The method of claim 1 , wherein step (c) and/or (d) are performed at a pH of between about 4.5 and about 7.0.

10. The method of claim 1 , wherein the support is provided in organic solvent prior to use in step (c).

11. The method of claim 1 , wherein the nucleic acid is provided in organic solvent prior to use in step (d).

12. The method of claim 1 , wherein the nucleic acid is combined with the support after activating the support in step (c).

13. The method of claim 1 , wherein the method further comprises (e) separating the coupling solution of step (d) and the support and washing the support, optionally wherein the washing is performed with an aqueous solution or water.

14. The method of claim 13 , wherein the method further comprises (f) contacting the support with NaOH solution and/or heating the support.

15. The method of claim 2 , wherein the salt is dissolved in an organic solvent prior to the use in step (c).

16. The method of claim 2 , wherein the salt is LiCl or NaCl.

17. The method of claim 2 , wherein the salt is present in steps (c) and (d) at a concentration of at least about 0.1 M and less than about 1.0 M.

18. The method of claim 1 , wherein the nucleic acid is provided at a concentration of between about 10 and about 10,000 pmol/mg support.

19. The method of claim 1 , wherein the support is selected from the group consisting of: particles, spheres, microparticles, nanoparticles, and beads.

20. The method of claim 1 , wherein the support is monodisperse and/or magnetic.

Assignees

• Life Tech As
• Life Technologies Corp

Inventors

• Jobert Laure

Classifications

• C12N15/1013 Primary

  by using magnetic beads · CPC title

• C12N15/10

  Processes for the isolation, preparation or purification of DNA or RNA (chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34) · CPC title

• C07H21/04

  with deoxyribosyl as saccharide radical · CPC title

• C07H1/00

  Processes for the preparation of sugar derivatives · CPC title

• C12Q1/6834

  Enzymatic or biochemical coupling of nucleic acids to a solid phase · CPC title