DOUBLE-STRANDED NUCLEIC ACID, USE AND KIT THEREOF IN RIBONUCLEASE DETECTION

• US 20150037805A1
• Filed: 09/10/2012
• Published: 02/05/2015
• Est. Priority Date: 09/20/2011
• Status: Active Grant

• Current Assignee: Souzhou Ribo Life Science CO.,LTD
• Original Assignee: Souzhou Ribo Life Science CO.,LTD
• Inventors: Zhang, Hongyan, Huang, Weiyan
• Granted Patent: US 9,540,682 B2
• Time in Patent Office: Days
• US Class Current: 435/6.14
• CPC Class Codes: C12Q 1/34 involving hydrolase
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: G01N 2333/922 Ribonucleases (RNAses); Deoxyribonucleases (DNAses)
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/57496 involving intracellular compounds

Abstract

Provided are a double-stranded nucleic acid and use thereof in ribonuclease detection, and a ribonuclease detection method and use thereof in tumor detection and/or diagnosis. Specifically, a double-stranded nucleic acid substrate comprises at least one ribonuclease sensitive site. The activity and content of the ribonuclease in a sample are detected by analyzing the degradation of the double-stranded nucleic acid substrate by the ribonuclease. Also provided are a ribonuclease detection kit and a tumor detection kit.

Claims

1. A double-stranded nucleic acid which can be cleaved by ribonuclease,

wherein the length of the double-stranded nucleic acid is 7-30 base pairs,

the base sequence of the first single strand of the double-stranded nucleic acid contains at least one CA base sequence or UA base sequence, the base sequence of the second single strand of the double-stranded nucleic acid contains UG base sequence or UA base sequence which is complementary to the CA base sequence or the UA base sequence in the first single strand,

one end of the double-stranded nucleic acid is attached to an fluorescence donor group, the other end the double-stranded nucleic acid is attached to an fluorescence acceptor group, and energy transfer can occur between the fluorescence donor group and the fluorescence acceptor group.

2. The double-stranded nucleic acid according to claim 1, wherein the length of the double-stranded nucleic acid is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 base pairs.

7. The double-stranded nucleic acid according to claim 1, wherein the first single strand and the second single strand of the double-stranded nucleic acid are perfectly complementary, and the base sequence of the first single strand is 5′

-AUGAGCCUGAUUU, 5′

-AUGAGCCUAAUUU, 5′

-GGCUGCU, 5′

-GAAUGAGCU, 5′

-GAGUCAGCUAATCUU or 5′

-UGGUAUGAGCCUGAUUUUGAU.

9. The double-stranded nucleic acid according to claim 1, wherein the double-stranded nucleic acid contains at least one deoxyribonucleotide group.

10. The double-stranded nucleic acid according to claim 1, wherein the double-stranded nucleic acid contains at least one modified nucleotide group.

11. The double-stranded nucleic acid according to claim 10, wherein the modified nucleotide group is a nucleotide group in which at least one of phosphate group, ribose group and base is modified.

12. The double-stranded nucleic acid according to claim 11, wherein the nucleotide group in which ribose group is modified is a nucleotide group in which 2′

-hydroxy of the ribose group is modified.

13. The double-stranded nucleic acid according to claim 12, wherein the nucleotide group in which ribose group is modified is a nucleotide group in which 2′

-hydroxy of the ribose group is substituted by methoxy or fluorine.

14. The double-stranded nucleic acid according to claim 1, wherein the fluorescence donor group and the fluorescence acceptor group are located on the same nucleic acid single strand.

15. The double-stranded nucleic acid according to claim 1, wherein the fluorescence donor group and the fluorescence acceptor group are located on different nucleic acid single strands.

17. The double-stranded nucleic acid according to claim 1, wherein the fluorescence acceptor group is a fluorescence quenching group.

18. The double-stranded nucleic acid according to claim 1, wherein the fluorescence donor group is at least one selected from the group consisting of fluorescein group, tetrachlorofluorescein group, hexachlorofluorescein group, rhodamine group, tetramethylrhodamine group, Cy dye group, Texas Red group, Bodipy dye group and Alexa dye group;

preferably, the fluorescence donor group is at least one selected from the group consisting of 6-carboxyfluorescein group, 5-tetrachlorofluorescein group, 5-hexachlorofluorescein group, 6-carboxy-x-rhodamine group, indodicarbocyanine group and 6-carboxytetramethylrhodamine group.

20. The double-stranded nucleic acid according to claim 1, wherein the fluorescence acceptor group is at least one selected from the group consisting of nitrogen-substituted xanthene group, 4-(phenyldiazenyl)phenylamine group and 4-(phenyldiazenyl) naphthylamine groups;

preferably, the fluorescence acceptor group is at least one selected from the group consisting of 4-(4′

-dimethylaminophenylazo)benzoic acid group, N,N′

-dimethyl-N,N′

-diphenyl-4-((5-t-butoxycarbonylaminopentyl)aminocarbonyl)piperidinylsulfonerhodamine group, 4′

,5′

-dinitrofluorescein group, pipecolic acid amide group, 4-[4-nitrophenyldiazinyl]phenylamine group and 4-[4-nitrophenyldiazinyl]naphthylamine group.

22. Use of the double-stranded nucleic acid according to claim 1 in ribonuclease detection.

23. The use according to claim 22, wherein the ribonuclease is at least one selected from the group consisting of RNase I, RNase A and RNase H.

24. Use of the double-stranded nucleic acid according claim 1 in the manufacture of ribonuclease detection reagent.

25. The use according to claim 24, wherein the ribonuclease is at least one selected from the group consisting of RNase I, RNase A and RNase H.

26. A ribonuclease detection method, including the following steps:

1) obtaining the double-stranded nucleic acid according to claim 1;

2) contacting the double-stranded nucleic acid in step

     1) with a test sample, such that the double-stranded nucleic acid can be cleaved by the ribonuclease possibly existing in the test sample;

3) detecting a product after said contacting in step

     2), thereby detecting the ribonuclease in the test sample, or

detecting the amount of a cleavage product in the product after said contacting in step

     2), thereby detecting the content of the ribonuclease in the test sample,

wherein the cleavage product or the amount thereof is detected by measuring a fluorescence signal with a specific wavelength emitted by the fluorescence donor group and/or the fluorescence acceptor group attached to both ends of the double-stranded nucleic acid.

29. The detection method according to claim 26, wherein the test sample is at least one selected from the group consisting of blood, plasma, serum, saliva, tissue fluid and urine sample.

30. Use of the detection method in claim 26 in tumor detection and/or diagnosis, wherein the tumor is gastric cancer, colon cancer or lung cancer.

32. A ribonuclease detection kit, containing an effective amount of the double-stranded nucleic acid according to claim 1.

33. A tumor detection kit, characterized in that the kit contains the double-stranded nucleic acid according to claim 1.

3-6. -6. (canceled)

8. (canceled)

16. (canceled)

19. (canceled)

21. (canceled)

27-28. -28. (canceled)

31. (canceled)

34. (canceled)