Methods of functionalization and reagents used in such methods using an aza-isatoic anhydride or a derivative thereof, biological molecules thus treated and kits

• US 10,174,068 B2
• Filed: 07/26/2013
• Issued: 01/08/2019
• Est. Priority Date: 08/02/2012
• Status: Active Grant

• Current Assignee: Biomerieux B.V. (Compagnie Merieux Alliance SAS)
• Original Assignee: Biomerieux B.V. (Compagnie Merieux Alliance SAS)
• Inventors: Cailly, Thomas, Fabis, Frederic, Laayoun, Ali, Laurent, Alain, Ursuegui, Sylvain
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US14/416,996
• Publication Number:
  US 20150210732A1
• Time in Patent Office: 1,992 Days
• Field of Search: 536 251
• CPC Class Codes: C07D 213/80 in position 3
• CPC Class Codes: C07D 405/02 containing two hetero rings
• CPC Class Codes: C07D 495/04 Ortho-condensed systems
• CPC Class Codes: C07D 519/00 Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00
• CPC Class Codes: C07H 1/00 Processes for the preparation of sugar derivatives
• CPC Class Codes: C07H 21/02 with ribosyl as saccharide radical
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors

Abstract

The present invention relates to a method of functionalization of at least one ribonucleic acid (RNA) molecule, contained in a liquid sample, which includes the following steps: a) providing at least: one binding molecule consisting of an aza-isatoic anhydride or a derivative thereof, one group of interest, and one linkage joining the binding molecule to the group of interest, b) reacting the anhydride function of the binding molecule with at least one hydroxyl group carried: in position 2′ of the ribose of one of the RNA nucleotides, and/or in position(s) 2′ and/or 3′ of the ribose of the nucleotide at the terminal 3′ end of the RNA, and obtaining an aza-anthranilate that joins, by means of the linkage, the RNA to the group of interest.

Claims

1. A functionalizing reagent of formula (I):

2. The functionalizing reagent according to claim 1, wherein Y represents Z⁴ so that the functionalizing reagent has the following formula (1):

3. The functionalizing reagent according to claim 1, wherein Y represents the group C—

X³—

R³ so that the functionalizing reagent has the following formula (2);

4. The functionalizing reagent according to claim 1, wherein the functionalizing reagent is immobilized on a solid support.

5. The functionalizing reagent according to claim 1, wherein each of the linkages X¹, X² and X³ represents, independently of one another, a covalent bond or an optionally substituted carbon group having one or more carbon atoms and optionally containing an aromatic structure and/or heteroatom.

6. The functionalizing reagent according to claim 1, wherein one or more of the linkages X¹, X² and X³ comprise a bond or function capable of being cleaved in a physicochemical, photochemical, thermal, enzymatic and/or chemical manner.

7. The functionalizing reagent according to claim 1, wherein the group of interest is the marker or labelling precursor.

8. The functionalizing reagent according to claim 1, wherein the group of interest is a marker having an intrinsic fluorescence.

9. The functionalizing reagent according to claim 1, wherein the group of interest is a marker not having an intrinsic fluorescence.

10. A functionalized RNA comprising RNA functionalized with the functionalizing reagent according to claim 1.

11. A kit comprising the functionalizing reagent according to claim 1.

12. A method of functionalizing RNA contained in a liquid sample, comprising:

reacting an anhydride function of the functionalizing reagent according to claim 1 with at least one hydroxyl group in any of (i) position 2′

of riboses of nucleotides of RNA molecules, (ii) position 2′

of riboses of nucleotides at terminal 3′

ends of RNA molecules, or (iii) position 3′

of riboses of nucleotides at terminal 3′

ends of RNA molecules.

13. The method according to claim 12, wherein the group of interest has an intrinsic fluorescence.

14. The method according to claim 12, wherein the group of interest does not have an intrinsic fluorescence.

15. The method according to claim 12, wherein the group of interest is a ligand complementary to an anti-ligand and the method further comprises:

capturing the functionalized RNA by binding the ligand with the anti-ligand.

16. The method according to claim 15, further comprising removing the captured RNA to obtain a DNA-enriched sample.

17. A method of separating RNA relative to other biological constituents including DNA, comprising:

obtaining a functionalized RNA by performing the method according to claim 12 in a biological sample containing RNA and DNA wherein the group of interest is immobilized on a solid support that is able to be separated from the biological sample; and

separating the functionalized RNA from the biological sample.

18. The method according to claim 12, wherein the anhydride function of the functionalizing reagent is reacted with the at least one hydroxyl group at room temperature.

19. The method according to claim 17, wherein the functionalized RNA is obtained by performing the method at room temperature.