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

• US 6,376,179 B1
• Filed: 12/17/1999
• Issued: 04/23/2002
• Est. Priority Date: 06/17/1998
• Status: Expired due to Term

• Current Assignee: Bio Merieux
• Original Assignee: Bio Merieux
• Inventors: Laayoun, Ali
• Primary Examiner(s): Fredman, Jeffrey
• Assistant Examiner(s): CHAKRABARTI, ARUN K
• Application Number:
  US09/446,156
• Time in Patent Office: 858 Days
• Field of Search: 435/6, 435/91.2, 536/25.3, 536/25.31, 536/25.32, 540/474, 540/465, 556/1, 556/6
• US Class Current: 435/5
• 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

Abstract

The present invention relates to a process for labeling a synthetic or natural ribonucleic acid (RNA). It also relates to RNA fragments, which have been labeled by fragmenting the RNA to free a terminal phosphate of each fragment for further reaction, and labeling each fragment at the freed terminal phosphate which is located at the 3′ end and/or the 5′ end of each fragment of the RNA, and to the use of such RNA fragments, for example, in the field of medical diagnosis.

Claims

1. A process for labeling a synthetic or natural ribonucleic acid (RNA), comprising:

fragmenting the RNA to produce a plurality of RNA fragments having freed terminal phosphates for further reaction, and

labeling a plurality of said fragments at the terminal phosphates freed in the fragmentation step, located at the 3′

end and/or the 5′

end of said fragments.

2. The process according to claim 1, wherein the fragmentation and the labeling are effected in two steps.

3. The process according to claim 1, wherein the labeling of the 3′

end or the 5′

end of an RNA fragment is effected by binding a binding agent having a reactive function, which is linked to a label, or which is capable of being subsequently linked to a label, to the phosphate that is in the 2′

position, in the 3′

position or in the cyclic monophosphate 2′

-3′

position, with respect to the ribose.

4. The process according to claim 1, wherein the fragmentation and/or the labeling of the 3′

end or the 5′

end of an RNA fragment is effected by binding a binding agent having a nucleophilic, electrophilic or halide function, which is linked to a label, or which is capable of being subsequently linked to at least one label, to the phosphate in the 2′

position, in the 3′

position or in the cyclic monophosphate 2′

-3′

position, with respect to the ribose.

5. The process according to claim 1, wherein the fragmentation of the RNA is effected enzymically, chemically or physically.

6. The process according to claim 5, wherein the enzymic fragmentation of the RNA is carried out by means of nucleases.

7. The process according to claim 5, wherein the chemical fragmentation of the RNA is carried out by means of metal cations.

8. The process according to claim 5, wherein the physical fragmentation of the RNA is carried out by means of sonication or by means of radiation.

9. The process according to claim 1, wherein the labeling of the 3′

end or the 5′

end of an RNA fragment is effected by binding a molecule R—

X, where R consists of the label and X is the agent for binding the label to the RNA, to the phosphate, which is linked to the 2′

position, to the 3′

position or to the cyclic monophosphate 2′

-3′

position of the ribose.

10. An RNA fragment that is obtained by the process according to claim 1, wherein the RNA fragment comprises,

a single nucleotide which is labeled at the freed terminal phosphate that is located at the 3′ end or the 5′

end of the RNA fragment, and

at least one other nucleotide whose base (purine;

adenine/guanine or pyrimidine;

uracil/cytosine) is identical to that of the labeled nucleotide,

wherein the fragment comprises from 10 to 100 nucleotides.

11. An RNA fragment according to claim 10, wherein the RNA fragment comprises at least one thiophosphate nucleotide.

12. An RNA fragment according to claim 11, wherein the labeled nucleotide is a thiophosphate nucleotide.

13. An RNA fragment according to claim 10, wherein the fragment comprises from 30 to 70 nucleotides.

14. An RNA fragment according to claim 10, wherein the fragment comprises from 40 to 60 nucleotides.

15. The process according to claim 1, wherein the 3′

end and/or the 5′

end of each fragment is labeled, except that the original 3′

end and the original 5′

end of the starting RNA are not labeled.

16. The process of claim 7, wherein said metal cations are combined with a chemical catalyst.

17. The process according to claim 16, wherein the metal cations are Mg⁺⁺, Mn⁺⁺, Cu⁺⁺, Co⁺⁺ and/or Zn⁺⁺ ions and the chemical catalyst is an imidazole, a substituted analogue, or any chemical molecule that has an affinity for the RNA and that carries an imidazole nucleus or a substituted analogue.

18. The process according to claim 17, wherein the chemical catalyst is N-methylimidazole.

19. The process according to claim 9, wherein the agent for binding the label to the RNA is at least one member selected from the group consisting of hydroxyl, amine, hydrazine, alkoxylamine, alkyl halide, phenylmethyl halide, iodoacetamide and a maleimide grouping.

20. A method for detecting at least one nucleic acid selected from the group consisting of a DNA, a DNA fragment, an RNA, and an RNA fragment, comprising:

obtaining a sample containing said nucleic acid, and

contacting said sample with an RNA fragment probe according to claim 13 to detect for said nucleic acid.

21. A method for binding a capture probe with a labeled target, comprising:

obtaining a sample containing said capture probe, and

contacting said sample with a labeled RNA fragment according to claim 13, which is able to bind to said capture probe.

22. A process for labeling a synthetic or natural ribonucleic acid (RNA), comprising:

fragmenting the RNA to produce a plurality of RNA fragments having freed terminal phosphates for further reaction, and

labeling with a label a plurality of said fragments at the terminal phosphates freed in the fragmentation step, located at the 3′

end and/or the 5′

end of said fragments, wherein the label is directly detectable.

23. The process of claim 22, wherein said label is horseradish peroxidase, alkaline phosphatase, β

-galactosidase or glucose-6-phosphate dehydrogenase.

24. The process of claim 22, wherein said label is a fluorescent or luminescent compound or dye.

25. The process of claim 22, wherein said label is ³²P, ³⁵S or ¹²⁵I.

34. The process of claim 22, wherein the label comprises at least one enzyme that produces a detectable signal.

35. The process of claim 22, wherein the label comprises at least one chromophore.

36. The process of claim 22, wherein the label comprises at least one radioactive molecule.

26. A process for labeling a synthetic or natural ribonucleic acid (RNA), comprising:

fragmenting the RNA to produce a plurality of RNA fragments having freed terminal phosphates for further reaction, and

labeling with a label a plurality of said fragments at the terminal phosphates freed in the fragmentation step, located at the 3′

end and/or the 5′

end of said fragments, wherein the label is detectable by a physical, chemical or electrical characteristic of the label.

27. The process of claim 26, wherein the label is detectable by at least one of electron density, conductivity, amperometry, voltametry and impedance of the label.

28. The process of claim 26, wherein the label is detectable by diffraction, surface plasmon resonance, surface variation and angle of contact variation.

29. The process of claim 26, wherein the label is detectable by atomic force spectroscopy and the tunnel effect.

30. A process for labeling a synthetic or natural ribonucleic acid (RNA), comprising:

fragmenting the RNA to produce a plurality of RNA fragments having freed terminal phosphates for further reaction, and

labeling a plurality of said fragments at said freed terminal phosphates located at the 3′

end and/or the 5′

end of the fragments,

wherein the fragmentation and the labeling are effected in one step.

31. A process for labeling a natural ribonucleic acid (RNA), comprising:

fragmenting the natural RNA to produce a plurality of RNA fragments having freed terminal phosphates for further reaction, and

labeling a plurality of said fragments at freed terminal phosphates located at the 3′

end and/or the 5′

end of said fragments.

32. The process of claim 31, wherein the fragmentation and the labeling are effected in at least two steps.

33. A process for labeling a synthetic or natural ribonucleic acid (RNA), comprising:

non-specifically fragmenting the RNA to produce a plurality of RNA fragments having freed terminal phosphates for further reaction, and

labeling a plurality of said fragments at freed terminal phosphates located at the 3′

end and/or the 5′

end of said fragments.