Process for labeling a ribonucleic acid

• US 6,921,640 B2
• Filed: 03/20/2002
• Issued: 07/26/2005
• Est. Priority Date: 06/17/1998
• Status: Expired due to Fees

• Current Assignee: Bio Merieux
• Original Assignee: Bio Merieux
• Inventors: Laayoun, Ali
• Primary Examiner(s): RILEY, JEZIA
• Assistant Examiner(s): Chakrabarti, Arun
• Application Number:
  US10/101,362
• Publication Number:
  US 20020187500A1
• Time in Patent Office: 1,224 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/24.3, 536/25.3, 536/25.31, 536/25.32, 935/6, 436/518, 540/465, 540/474, 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:

obtaining prepared RNA by a method selected from the group consisting of;

amplifying the RNA;

amplifying a template deoxyribonucleic acid (DNA) into the RNA; and

amplifying a template DNA of the RNA and transcribing the resulting amplified DNA into the RNA;

fragmenting the prepared RNA to produce a plurality of RNA fragments having freed terminal phosphates located at at least one of the 3′ and

5′

ends of the RNA fragments; and

labeling a plurality of the RNA fragments at terminal phosphates freed by fragmenting.

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

3. The process according to claim 1, wherein:

the labeling is effected by binding a binding agent having a reactive function to a phosphate;

the binding agent being linked to a label or being capable of being subsequently linked to a label; and

the phosphate being in a 2′

position, a 3′

position or a cyclic monophosphate 2′

-3′

position, with respect to ribose.

4. The process according to claim 1, wherein:

at least one of the fragmenting and the labeling is effected by binding a binding agent to a phosphate;

the binding agent having a nucleophilic, electrophilic or halide function;

the binding agent being linked to a label or being capable of being subsequently linked to at least one label; and

the phosphate being in a 2′

position, a 3′

position or a cyclic monophosphate 2′

-3′

position, with respect to ribose.

5. The process according to claim 1, wherein the fragmenting comprises enzymatic, chemical or physical fragmentation.

6. The process according to claim 5, wherein the enzymatic fragmentation is carried out using nucleases.

7. The process according to claim 5, wherein the chemical fragmentation is carried out using metal cations.

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

9. The process according to claim 8, wherein:

the metal cations comprise at least one member selected from the group consisting of Mg⁺⁺, Mn⁺⁺, Cu⁺⁺, Co⁺⁺ and Zn⁺⁺ ions; and

the chemical catalyst comprises at least one member selected from the group consisting of imidazole, a substituted analogue and a chemical molecule, the chemical molecule having an affinity for the RNA and carrying an imidazole nucleus or a substituted analogue.

10. The process according to claim 9, wherein the chemical catalyst comprises N-methylimidazole.

11. The process according to claim 5, wherein the physical fragmentation is carried out using sonication or radiation.

12. The process according to claim 1, wherein:

the labeling is effected by binding molecule R-X to the phosphate;

R being a label and X being an agent for binding the label to the RNA; and

the phosphate being linked to a 2′

position, a 3′

position or a cyclic monophosphate 2′

-3′

position of a ribose.

13. The process according to claim 12, wherein X is at least one member selected from the group consisting of hydroxyl, amine, hydrazine, alkoxylamine, alkyl halide, phenylmethyl halide, iodoacetamide and malemide groups.

14. The process according to claim 1, wherein:

at least one of the 3′ and

5′

ends of each fragment is labeled; and

3′ and

5′

ends of the prepared RNA are not labeled.

15. The process according to claim 1, wherein the fragmentation and the labeling are effected in one step.

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

obtaining extracted RNA by extraction from cell material;

fragmenting the extracted RNA to produce a plurality of RNA fragments having freed terminal phosphates at at least one of 3′ and

5′

ends of the fragments, and

labeling a plurality of the RNA fragments at terminal phosphates freed by fragmenting.

17. The process of claim 16, comprising amplifying the extracted RNA before fragmenting.

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

obtaining transcribed RNA by;

extracting a template deoxyribonucleic acid (DNA) of the RNA from cell material and transcribing the extracted DNA into RNA;

fragmenting the transcribed RNA to produce a plurality of RNA fragments having freed terminal phosphates located at at least one of the 3′ and

5′

ends of the fragments; and

labeling a plurality of the RNA fragments at terminal phosphates freed by fragmenting.

19. The process of claim 18, comprising amplifying the transcribed RNA before fragmenting.

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

obtaining a template deoxyribonucleic acid (DNA) of RNA by extraction from cell material;

amplifying the DNA into the RNA;

fragmenting the amplified RNA to produce a plurality of RNA fragments having freed terminal phosphates located at at least one of the 3′ and

5′

ends of the fragments; and

labeling a plurality of the RNA fragments at terminal phosphates freed by fragmenting.