Hybrid interfering rna

Abstract

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 hybrid nucleic acid molecule comprising an inhibitory double stranded ribonucleic acid (RNA) operably linked to a single stranded deoxyribonucleic acid (DNA) primer, wherein the primer anneals to a nucleic acid template and is extendable in a polymerase chain reaction. 
     
     
         2 . The hybrid nucleic acid molecule of  claim 1 , wherein the 5′ end of the single stranded DNA primer is linked to the 5′ end of the sense strand or the antisense strand of the double stranded inhibitory RNA molecule. 
     
     
         3 . The hybrid nucleic acid molecule of  claim 1 , wherein the 5′ end of the single stranded DNA primer is linked to the 3′ end of the sense strand or the antisense strand of the double stranded inhibitory RNA molecule. 
     
     
         4 . The hybrid nucleic acid molecule of  claim 1 , wherein the 3′ end of the single stranded DNA primer is linked to the 5′ end of the sense strand or the antisense strand of the double stranded inhibitory RNA molecule. 
     
     
         5 . The hybrid nucleic acid molecule of  claim 1 , wherein the 3′ end of the single stranded DNA primer is linked to the 3′ end of the sense strand or the antisense strand of the double stranded inhibitory RNA molecule. 
     
     
         6 . The hybrid nucleic acid molecule of  claim 1 , wherein the DNA primer is linked to the double stranded inhibitory RNA molecule covalently. 
     
     
         7 . The hybrid nucleic acid molecule of  claim 1 , wherein the DNA primer is at least 7 nucleotides in length. 
     
     
         8 . The hybrid nucleic acid molecule of  claim 1 , wherein the double stranded inhibitory RNA molecule is at least 18 base pairs in length. 
     
     
         9 . The hybrid nucleic acid molecule of  claim 1 , wherein the sense strand of the double stranded inhibitory RNA molecule encodes at least 18 nucleotides of at least one mRNA. 
     
     
         10 . A method of inhibiting expression of a gene in a subject comprising administering to the subject a pharmaceutical composition comprising a hybrid nucleic acid molecule specific for an mRNA encoded by the gene in an amount effective to reduce expression of the gene compared to a control,
 wherein the hybrid nucleic acid molecule comprises an inhibitory double stranded ribonucleic acid (RNA) operably linked to a single stranded primer,   wherein the primer anneals to a nucleic acid template and is extendable in a polymerase chain reaction.   
     
     
         11 . The method of  claim 10  wherein the primer is a DNA primer. 
     
     
         12 . The method of  claim 10  wherein the sequence of the nucleic acid template is predetermined and the sequence of the primer is complementary to the sequence of the template. 
     
     
         13 . The method of  claim 10 , wherein the gene is a disease associated gene. 
     
     
         14 . The method of  claim 10 , wherein the gene is a viral gene. 
     
     
         15 . The method  claim 14 , wherein the viral gene is E6 or E7 of human papilloma virus. 
     
     
         16 . The method of  claim 13 , wherein the subject has a disease caused by the disease associated gene. 
     
     
         17 . The method of  claim 13 , wherein the subject has cancer. 
     
     
         18 . The method of  claim 17 , wherein the cancer is selected from the group consisting of cervical cancer, prostate cancer, breast cancer, melanoma, hepatoma, renal cancer, glioma, bladder cancer, lung cancer, cancer of the central nervous system, ovarian cancer, testicular cancer, cancer of the pancreas, gastrointestinal cancer, lymphoma, seminoma, embryonal carcinoma, yolk sac carcinoma, choriocarcinoma, and osteocarcinoma. 
     
     
         19 . A method for detecting the presence of a hybrid nucleic acid molecule comprising detecting the presence of a PCR-amplification product of a nucleic acid template in a PCR preparation comprising a cell extract suspected of comprising the hybrid nucleic acid molecule, wherein the hybrid nucleic acid molecule comprises an inhibitory double stranded ribonucleic acid (RNA) operably linked to a PCR primer, wherein the PCR primer anneals to the nucleic acid template, and wherein the presence of the PCR-amplification product is indicative of the presence of the hybrid nucleic acid molecule. 
     
     
         20 . The method of  claim 19  wherein the PCR preparation further comprises a second primer. 
     
     
         21 . 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 the single stranded DNA molecule is covalently bound to the 3′ end of the anti-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).