Novel rna interference methods using dna-rna duplex constructs

Abstract

The present invention provides novel compositions and methods for suppressing the function or activity of a targeted gene through a novel intracellular piRNA-mediated RNAi mechanism, using RNA-DNA duplex constructs. The invention further provides novel methods and compositions for generating or producing RNA-DNA duplex agents, whose quantity is high enough to be used for the invention's gene silencing transfection and possibly in therapeutics applications. This improved RNA-polymerase chain reaction (RNA-PCR) method utilizes thermocycling steps of promoter-linked DNA or RNA template synthesis, in vitro transcription and then reverse transcription to bring up the amount of RNA-DNA duplexes up to two thousand folds within one round of the above procedure for using in D-RNAi-directed gene silencing.

1 . A method for inhibiting a targeted gene function or activity through an intracellular RNA interference mechanism in a cell or organism that expresses the targeted gene, comprising the steps of:
 a) providing a composition comprising an RNA-DNA duplex agent prior to contacting said cell or organism, wherein the RNA-DNA duplex agent is capable of inhibiting the function and/or expression activity of said targeted gene in said cell and organism; and   b) contacting said cell with said composition under conditions such that the function and/or activity of said gene in said cell or organism is inhibited.   wherein the RNA is a ribonucleic acid sequence containing high complementarity to said targeted gene and the DNA is a deoxyribonucleic acid sequence complementary to said RNA, and wherein the RNA-DNA duplex is formed by hybridization between said RNA and said DNA in a sequence complementary region containing more than 24 base pairs.   
     
     
         2 . The method of  claim 1 , wherein said cell or organism expresses said targeted gene in vitro. 
     
     
         3 . The method of  claim 1 , wherein said cell or organism expresses said targeted gene in vivo. 
     
     
         4 . The method of  claim 1 , wherein said targeted gene comprises a gene selected from the group consisting of pathogenic nucleic acids, viral genes, mutated genes, oncogenes, functional cellular genes, jumping genes, and transposon. 
     
     
         5 . The method of  claim 1 , wherein said RNA-DNA duplex agent inhibits a gene selected from the group consisting of bcl-2, beta-catenin, sonic hedgehog, tyrosinase, and HIV. 
     
     
         6 . The method of  claim 1 , wherein said cell is a eukaryotic cell. 
     
     
         7 . The method of  claim 1 , wherein said organism is a eukaryote. 
     
     
         8 . The method of  claim 7 , wherein said eukaryote is a vertebrate. 
     
     
         9 . The method of  claim 7 , wherein said eukaryote is a chicken. 
     
     
         10 . The method of  claim 7 , wherein said eukaryote is a mouse. 
     
     
         11 . The method of  claim 7 , wherein said eukaryote is a chimpanzee. 
     
     
         12 . The method of  claim 7 , wherein said eukaryote is a human being. 
     
     
         13 . The method of  claim 1 , wherein the composition consists of at least an RNA-DNA duplex capable of inhibiting the function and/or expression activity of said targeted gene in said cell or organism through the activation of an intracellular mechanism similar to RNA interference. 
     
     
         14 . The method of  claim 13 , wherein said cell or organism expresses said targeted gene in vitro. 
     
     
         15 . The method of  claim 13 , wherein said cell or organism expresses said targeted gene in vivo. 
     
     
         16 . The method of  claim 13 , wherein said targeted gene comprises a gene selected from the group consisting of group consisting of pathogenic nucleic acids, viral genes, mutated genes, oncogenes, functional cellular genes, jumping genes, and transposon. 
     
     
         17 . The method of  claim 13 , wherein said RNA-DNA duplex inhibits a gene selected from the group consisting of bcl-2, beta-catenin, sonic hedgehog, tyrosinase, and HIV. 
     
     
         18 . The method of  claim 13 , wherein said cell is a eukaryotic cell. 
     
     
         19 . The method of  claim 13 , wherein said organism is a eukaryote. 
     
     
         20 . The method of  claim 19 , wherein said eukaryote is a vertebrate. 
     
     
         21 . The method of  claim 19 , wherein said eukaryote is a chicken. 
     
     
         22 . The method of  claim 19 , wherein said eukaryote is a mouse. 
     
     
         23 . The method of  claim 19 , wherein said eukaryote is a chimpanzee. 
     
     
         24 . The method of  claim 19 , wherein said eukaryote is a human being. 
     
     
         25 . The method of  claim 13 , wherein said RNA-DNA duplex is formed by enzymatic reactions of in-vitro transcription and reverse transcription. 
     
     
         26 . The method of  claim 13 , wherein said RNA-DNA duplex is formed by chemical synthesis and hybridization. 
     
     
         27 . A method for inhibiting a targeted gene function or expression activity in a cell or organism that expresses the targeted gene, comprising the steps of:
 a) providing a composition comprising an RNA-DNA hybrid duplex agent prior to contacting said cell or organism, wherein the RNA-DNA hybrid duplex agent is capable of inhibiting the function and/or expression activity of said targeted gene in said cell and organism; and   b) contacting said cell with said composition under conditions such that the function and/or expression activity of said gene in said cell or organism is inhibited.   
     
     
         28 . The method of  claim 27 , wherein said cell or organism expresses said targeted gene in vitro. 
     
     
         29 . The method of  claim 27 , wherein said cell or organism expresses said targeted gene in vivo. 
     
     
         30 . The method of  claim 27 , wherein said targeted gene comprises a gene selected from the group consisting of pathogenic nucleic acids, viral genes, mutated genes, oncogenes, functional cellular genes, jumping genes, and transposon. 
     
     
         31 . The method of  claim 27 , wherein said RNA-DNA hybrid duplex agent inhibits a gene selected from the group consisting of bcl-2, beta-catenin, sonic hedgehog, tyrosinase, and HIV. 
     
     
         32 . The method of  claim 27 , wherein said cell is a eukaryotic cell. 
     
     
         33 . The method of  claim 27 , wherein said organism is a eukaryote. 
     
     
         34 . The method of  claim 33 , wherein said eukaryote is a vertebrate selected from the group consisting of chicken, mouse, chimpanzee, and human being. 
     
     
         35 . The method of  claim 27 , wherein the composition consists of at least an RNA-DNA hybrid duplex capable of inhibiting the function and/or expression activity of said targeted gene in said cell or organism. 
     
     
         36 . The method of  claim 35 , wherein said cell or organism expresses said targeted gene in vitro. 
     
     
         37 . The method of  claim 35 , wherein said cell or organism expresses said targeted gene in vivo. 
     
     
         38 . The method of  claim 35 , wherein said targeted gene comprises a gene selected from the group consisting of pathogenic nucleic acids, viral genes, mutated genes, oncogenes, functional cellular genes, jumping genes, and transposon. 
     
     
         39 . The method of  claim 35 , wherein said RNA-DNA hybrid duplex agent inhibits a gene selected from the group consisting of bcl-2, beta-catenin, sonic hedgehog, tyrosinase, and HIV. 
     
     
         40 . The method of  claim 35 , wherein said cell is a eukaryotic cell. 
     
     
         41 . The method of  claim 35 , wherein said organism is a eukaryote. 
     
     
         42 . The method of  claim 41 , wherein said eukaryote is a vertebrate selected from the group consisting of chicken, miouse, chimpanzee, and human being. 
     
     
         43 . The method of  claim 35 , wherein said RNA-DNA hybrid duplex is formed by enzymatic reactions of in-vitro transcription and reverse transcription. 
     
     
         44 . The method of  claim 35 , wherein said RNA-DNA hybrid duplex is formed by chemical synthesis and hybridization.