Process for labeling a ribonucleic acid, and labeled RNA fragments which are obtained thereby

• US 6,489,114 B2
• Filed: 12/18/2000
• Issued: 12/03/2002
• Est. Priority Date: 12/17/1999
• Status: Active Grant

• Current Assignee: Bio Merieux, Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Bio Merieux, Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Miyada, Charles G., Laayoun, Ali, Do, Duc
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): CHAKRABARTI, ARUN K
• Application Number:
  US09/737,761
• Publication Number:
  US 20010044105A1
• Time in Patent Office: 715 Days
• Field of Search: 435/6, 435/91.51, 435/235.1, 435/91.1, 435/91.2, 435/375, 540/465, 540/474, 536/24.2, 935/29, 935/39, 935/73
• US Class Current: 435/6.12
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin

Abstract

A process is provided for labeling with signal amplification a ribonucleic acid (RNA), comprising fragmenting the RNA to form RNA fragments, fixing a first ligand to a terminal phosphate located at least one of the 3′ end and the 5′ end of each of a plurality of the RNA fragments, the terminal phosphate having been released during the fragmentation, and binding a plurality of labeling agents to the first ligand on each of a plurality of the fragments.

Claims

1. A process for labeling with signal amplification a ribonucleic acid (RNA), comprising:

fragmenting the RNA to form RNA fragments,

fixing a first ligand to a terminal phosphate located at least one of the 3′

end and the 5′

end of each of a plurality of said RNA fragments, said terminal phosphate having been released during the fragmentation, and

binding a plurality of labeling agents to said first ligand on each of a plurality of said fragments.

2. A process according to claim 1, wherein the binding of the labeling agents to said first ligand is effected indirectly.

3. A process according to claim 2, wherein a first antiligand is bound to said first ligand, a second ligand is bound to said first antiligand, and the labeling agent comprises a second antiligand bearing at least one label and able to react with said second ligand.

4. A process according to claim 3, wherein first ligand/first antiligand and second ligand/second antiligand combinations are selected from the group consisting of biotin/streptavidin, hapten/antibody, antigen/antibody, peptide/antibody, sugar/lectin and polynucleotide/complementary polynucleotide.

5. A process according to claim 4, wherein the first and second ligands are the same.

6. A process according to claim 4, wherein the first and second ligands are different.

7. A process according to claim 6, wherein the first ligand is a derivative of fluorescein and the second ligand is a derivative of biotin.

8. A process according to claim 1, wherein the first ligand is a derivative of biotin and the labeling agent is a derivative of streptavidin.

9. A process according to claim 1, wherein the fragmenting and the fixing are effected in one step.

10. A process according to claim 1, wherein the fragmenting and the fixing are effected in two steps.

11. A process according to claim 1, wherein the binding of the labeling agent to the first ligand is covalent.

12. A process according to claim 1, wherein the binding of the labeling agent to the first ligand is non-covalent.

13. A process according to claim 1, wherein the fixing is effected by reacting a reactive function, which is carried by said first ligand, to a phosphate which is in the 2′

position, in the 3′

position or in the cyclic monophosphate 2′

-3′

position, with respect to a ribose at the 3′

end or the 5′

end of the RNA fragment.

14. A process according to claim 1, wherein at least one of the fragmenting and the fixing is effected by reacting a nucleophilic, electrophilic or halide function which is carried by said first ligand to a phosphate in the 2′

position, in the 3′

position or in the cyclic monophosphate 2′

-3′

position, with respect to a ribose at the 3′

end or the 5′

end of the RNA fragment.

15. A process according to claim 1, wherein the fragmenting is effected enzymatically, chemically or physically.

16. A process according to claim 15, wherein the fragmenting is carried out enzymatically with at least one nuclease.

17. A process according to claim 15, wherein the fragmenting is carried out chemically with metal cations optionally combined with a chemical catalyst.

18. A process according to claim 1, wherein the fragmenting is carried out with metal cations selected from the group consisting of Mg⁺⁺, Mn⁺⁺, Cu⁺⁺, Co⁺⁺ and Zn⁺⁺ ions, and a chemical catalyst comprised of imidazole, a substituted imidazole analogue, or any chemical molecule which has an affinity for the RNA and which carries an imidazole nucleus or a substituted imidazole analogue.

19. A process according to claim 15, wherein the fragmenting is carried out physically by sonication or irradiation.

20. A process according to claim 1, wherein the fixing is effected by reacting a molecule R-X to a phosphate which is linked to the 2′

position, to the 3′

position or to the cyclic monophosphate 2′

-3′

position of a ribose at the 3′

end or the 5′

end of the RNA fragment, where R is the first ligand and X is a reactive function selected from the group consisting of hydroxyl, amine, hydrazine, alkoxylamine, alkyl halide, phenylmethyl halide, iodoacetamide and maleimide.

21. A process according to claim 20, wherein R-X is selected from the group consisting of 5-(bromofluorescein) and derivatives of iodoacetyl biotin.

22. A method of detecting at least one member selected from the group consisting of an RNA, a DNA, an RNA fragment and a DNA fragment, comprising probing a sample suspected of containing said member with a labeled RNA fragment- obtained by the process of claim 1, wherein the RNA fragment comprises at the 3′

end or the 5′

end a single nucleotide which is labeled at the terminal phosphate released during the fragmentation.

24. A method of binding a labeled target to a capture probe, comprising exposing said labeled target to said capture probe, wherein said labeled target is a labeled RNA fragment obtained by the process of claim 1, wherein the RNA fragment comprises at the 3′

end or the 5′

end a single nucleotide which is labeled at the terminal phosphate released during the fragmentation.

23. A method of detecting at least one member selected from the group consisting of an RNA, a DNA, an RNA fragment and a DNA fragment, comprising probing a sample suspected of containing said member with a labeled RNA fragment-, the RNA fragment comprising at 3′

end a phosphate or a thiophosphate bearing a fluorescein bound to an anti-fluorescein antibody bearing at least one biotin, said antibody bound to a labeled streptavidin.

25. A method of binding a labeled target to a capture probe, comprising exposing said labeled target to said capture probe, wherein said labeled target is a labeled RNA fragment, the RNA fragment comprising at 3′

end a phosphate or a thiophosphate bearing a fluorescein bound to an anti-fluorescein antibody bearing at least one biotin, said antibody bound to a labeled streptavidin.