Hybrid interfering RNA
The invention relates to a hybrid interfering RNA molecule comprising a duplex RNA and a single stranded DNA molecule and its use in the ablation of mRNA and in polymerase chain reactions.
1. A nucleic acid molecule comprising
a first part that comprises a double stranded inhibitory ribonucleic acid (RNA) molecule comprising a sense strand and an antisense strand;
and a second part that comprises a single stranded deoxyribonucleic acid (DNA) molecule, wherein the 5′ end of said single stranded DNA molecule is covalently bound to the 3′ end of the sense strand of the double stranded inhibitory RNA molecule, and wherein the single stranded DNA molecule comprises a nucleotide sequence that anneals to a predetermined DNA template and functions as a primer to amplify the predetermined DNA template in a polymerase chain reaction (PCR).
2. The nucleic acid molecule according to claim 1 , wherein said single stranded DNA molecule comprises at least one copy of SEQ ID NO:1, wherein the 5′ end of SEQ ID NO:1 is contiguous with the 3′ end of the single stranded DNA molecule that functions as a primer.
3. The nucleic acid molecule according to claim 1 , wherein said single stranded DNA molecule is adapted to be resistant to nuclease digestion.
4. The nucleic acid molecule according to claim 1 , wherein said single stranded DNA molecule is between 10 and 40 nucleotides in length.
5. The nucleic acid molecule according to claim 1 , wherein said double stranded inhibitory RNA molecule is at least 19 base pairs in length.
6. The nucleic acid molecule according to claim 1 , wherein said double stranded inhibitory RNA molecule is between 18 and 29 base pairs in length.
7. The nucleic acid molecule according to claim 5 , wherein said double stranded inhibitory RNA molecule is about 21 base pairs in length.
8. The nucleic acid molecule according to claim 1 , wherein the sense strand of said double stranded inhibitory RNA molecule encodes at least 18 nucleotides of at least one mRNA.
9. The nucleic acid molecule according to claim 8 , wherein said mRNA is encoded by a disease associated gene.
10. The nucleic acid molecule according to claim 9 , wherein said disease associated gene is a viral gene.
11. The nucleic acid molecule according to claim 10 , wherein said viral gene is a viral oncogene.
12. The nucleic acid molecule according to claim 10 , wherein said viral gene is derived from a virus selected from the group consisting of: Human Immunodeficiency Virus, Human T Cell Leukaemia Virus, papilloma virus, papovavirus, rhinovirus, poliovirus, herpesvirus, adenovirus, Epstein Barr virus, influenza virus, hepatitis B virus and hepatitis C virus.
13. The nucleic acid molecule according to claim 12 , wherein said viral gene is derived from a papilloma virus.
14. The nucleic acid molecule according to claim 12 , wherein said viral gene is derived from a human papilloma virus.
15. The nucleic acid molecule according to claim 14 , wherein said human papilloma virus is selected from the group consisting of: HPV-2, HPV-6, HPV-11, HPV-16, HPV-18, HPV-31, HPV-33, HPV-52, HPV-54, HPV-56, HPV-5 and HPV-8.
16. The nucleic acid molecule according to claim 15 , wherein said human papilloma virus is derived from HPV-16.
17. The nucleic acid molecule according to claim 15 , wherein said viral gene is E6 or E7.
18. The nucleic acid molecule according to claim 1 , wherein said double stranded inhibitory RNA molecule comprises a modified base, sugar, internucleotide linkage, or combination thereof.
19. A pharmaceutical composition comprising the nucleic acid molecule according to claim 1 and a carrier.
20. An isolated cell transfected with the nucleic acid molecule according to claim 1 .
21. The nucleic acid molecule according to claim 17 , wherein said viral gene is E7.
22. The nucleic acid molecule according to claim 1 , wherein said single stranded DNA molecule is between 15 and 30 nucleotides in length.
23. The nucleic acid molecule according to claim 1 , wherein said double stranded inhibitory RNA molecule is between 21 and 27 base pairs in length.
24. The nucleic acid molecule according to claim 14 , wherein said single stranded DNA molecule is between 10 and 40 nucleotides in length.
25. The nucleic acid molecule according to claim 14 , wherein said single stranded DNA molecule is between 15 and 30 nucleotides in length.
26. The nucleic acid molecule according to claim 14 , wherein said double stranded inhibitory RNA molecule is between 21 and 27 base pairs in length.
27. The nucleic acid molecule according to claim 14 , wherein said double stranded inhibitory RNA molecule is at least 19 base pairs in length.
28. The nucleic acid molecule according to claim 14 , wherein said double stranded inhibitory RNA molecule is between 18 and 29 base pairs in length.
29. The nucleic acid molecule according to claim 14 , wherein said double stranded inhibitory RNA molecule is about 21 base pairs in length.
30. The nucleic acid molecule according to claim 14 , wherein said double stranded inhibitory RNA molecule comprises at least 18 base pairs in length.
31. The nucleic acid molecule according to claim 21 , wherein said single stranded DNA molecule comprises at least 10 nucleotides in length.
32. The nucleic acid molecule according to claim 21 , wherein the nucleotide sequence comprising the sense strand inhibitory RNA and the single stranded DNA comprises SEQ ID NO:15.
33. The nucleic acid molecule according to claim 21 , wherein the nucleotide sequence comprising the sense strand inhibitory RNA and the single stranded DNA consists of SEQ ID NO:15.
34. A pharmaceutical composition comprising the nucleic acid molecule according to claim 21 in an amount effective to reduce the expression of E7 mRNA and a pharmaceutically acceptable carrier.
35. The nucleic acid molecule according to claim 1 , wherein the double stranded inhibitory RNA molecule is a small interfering RNA (siRNA) molecule.
36. The nucleic acid molecule according to claim 14 , wherein the double stranded inhibitory RNA molecule is a small interfering RNA (siRNA) molecule.
37. A method for detecting the presence of a double stranded inhibitory RNA molecule in a sample comprising the steps of:
i) forming a preparation comprising the nucleic acid molecule of claim 1 , wherein the single stranded DNA present in the nucleic acid molecule of claim 1 is a first primer; a single stranded DNA molecule adapted to anneal to a predetermined DNA template and function as a second primer for a polymerase chain reaction (PCR); a predetermined DNA template; a thermostable DNA polymerase; and PCR components including nucleoside triphospahtes;
ii) providing reaction conditions that allow the amplification of said predetermined DNA template by the first primer and the second primer;
iii) and detecting the presence of a PCR-amplified product of the predetermined DNA template, wherein the presence of the PCR-amplified product of the predetermined DNA template correlates with the presence of the double stranded inhibitory RNA molecule in the sample.