Claims
- 1. A method for generating DNA-RNA hybrid constructs, comprising the steps of:
(a) providing: i) a solution comprising a nucleic acid template, ii) one or more primers sufficiently complementary to one oriented conformation of said nucleic acid template, and iii) one or more promoter-linked primers sufficiently complementary to the reversely oriented conformation of said nucleic acid template, and having an RNA promoter; (b) treating said nucleic acid template with said one or more primers under conditions such that a first DNA strand is synthesized; (c) treating said first DNA strand with said one or more promoter-linked primers under conditions such that a promoter-linked double-stranded nucleic acid is synthesized; (d) treating said promoter-linked double-stranded nucleic acid under conditions such that essentially amplified RNA fragments are synthesized; and (e) treating said RNA fragments with said one or more primers under conditions such that DNA-RNA hybrids are synthesized by reverse transcription of said amplified RNA fragments with the extension of said one or more primers.
- 2. The method of claim 1 further comprising the step of repeating steps b) through e) for a sufficient number of cycles to obtain a desired amount of amplified product.
- 3. The method of claim 1, wherein said treating step in step b) comprises heating said solution at a temperature above 90° C. to provide denatured nucleic acids.
- 4. The method of claim 1, wherein said treating step in step c) comprises pre-treating said first DNA strand with said one or more promoter-linked primers at a temperature ranging from about 35° C. to about 75° C.
- 5. The method of claim 1, wherein said treating step in step c) comprises treating said DNA strand with one or more promoter-linked primers in the presence of a polymerase.
- 6. The method of claim 5, wherein said polymerase is selected from the group consisting of DNA-dependent DNA polymerases, RNA-dependent DNA polymerases, RNA polymerases, Taq-like DNA polymerase, Tth-like DNA polymerase, C. therm. polymerase, viral replicases, and combinations thereof.
- 7. The method of claim 6, wherein said viral replicases are selected from the group consisting of avian myeloblastosis virus reverse transcriptase and Moloney murine leukemia virus reverse transcriptase, Brome mosaic virus replicase, Trichomonas vaginalis virus replicase, Flock house virus replicase, Q beta replicase, and mutants or combinations thereof.
- 8. The method of claim 7, wherein said avian myeloblastosis virus reverse transcriptase does not have RNase H activity.
- 9. The method of claim 1, wherein said treating step in step d) comprises treating said promoter-linked double-stranded nucleic acid with an enzyme having transcriptase activity at about 37° C.
- 10. The method of claim 9, wherein said enzyme having transcriptase activity is selected from the group consisting of T3 RNA polymerase, T7 RNA polymerase, SP6 RNA polymerase, M13 RNA polymerase and viral replicase.
- 11. The method as defined in claim 1, wherein said treating step in step e) comprises pre-treating said RNA fragments with said one or more primers at a temperature ranging from about 37° C. to about 72° C.
- 12. The method of claim 1, wherein said one or more promoter-linked primers are complementary to the 3′-ends of the antisense conformation of said nucleic acid template when said one or more primers are complementary to the 3′-ends of the sense conformation of said nucleic acid template.
- 13. The method of claim 1, wherein said one or more promoter-linked primers are complementary to the 3′-ends of the sense conformation of said nucleic acid template when said one or more primers are complementary to the 3′-ends of the antisense conformation of said nucleic acid template.
- 14. The method of claim 1, further comprising the step of generating one or more mismatched nucleotides in said DNA-RNA hybrid for gene silencing induction.
- 15. The method of claim 14, wherein said mismatched nucleotides are generated by enzymes selected from the group consisting of deaminase, Taq-like DNA polymerase, Tth-like DNA polymerase and viral replicases, or other low fidelity enzymes.
- 16. The method of claim 14, wherein said mismatched nucleotides are generated by chemical modification selected from the group consisting of weak acids, mild acetic anhydride and machine incorporation.
- 17. The method of claim 14, wherein said mismatched nucleotides are selected from the group consisting of deoxyuracil, inosine, xanthine, hypoxanthine, labeled nucleotide, ribonucleotide in a DNA construct, deoxyribonucleotide in an RNA construct, 7-deaza-dNTP, methylthio-linked nucleotide, phosphothio-linked nucleotide, morpholino nucleotide, peptide nucleic acid (PNA), and viral genome nucleic acid, etc.
- 18. The method of claim 1, further comprising the step of incorporating one or more nucleotide analogs into said DNA-RNA hybrid to increase gene silencing induction or to stabilize gene silencing effects.
- 19. The method of claim 18, wherein said nucleotide analogs are selected from the group consisting of deoxyuracil, labeled nucleotide, ribonucleotide in the DNA construct, deoxyribonucleotide in the RNA construct, 7-deaza-dNTP, methylthio-linked nucleotide, phosphothio-linked nucleotide, morpholino nucleotide, hexose-containing nucleotide, peptide nucleic acid (PNA) and their derivatives.
- 20. The method of claim 1, further comprising the step of contacting said DNA-RNA hybrid with a reagent for transfecting a eukaryotic cell for inhibiting the expression of at least one gene.
- 21. The method of claim 20, wherein said reagent is selected from the group consisting of chemical transfection reagents and liposomal transfection reagents.
- 22. The method as defined in claim 20, wherein said gene comprises a gene selected from the group consisting of pathogenic nucleic acids, viral genes, mutated genes, oncogenes and unknown functional genes.
- 23. A composition for inhibiting the expression of at least one targeted gene in a substrate, the composition comprising: a DNA-RNA hybrid.
- 24. The composition of claim 23, wherein the DNA-RNA hybrid is synthesized using the method of claim 1.
- 25. The composition of claim 23, wherein the RNA of said DNA-RNA hybrid is comprised of either part or all of the spliced mRNA transcript of the targeted gene.
- 26. The composition of claim 23, wherein the RNA of said DNA-RNA hybrid is either homologous or complementary to part or all of the unspliced mRNA transcript of the targeted gene.
- 27. The composition of claim 23, wherein the RNA of said DNA-RNA hybrid is either homologous or complementary to the combination of part or all of the unspliced and spliced mRNA transcript of the targeted gene.
- 28. The composition of claim 23, wherein the DNA-RNA hybrid is made by complementarily combining the RNA molecule of claims 25 or 26 with its corresponding complementary DNA molecule in a base-paring double-stranded form.
- 29. The composition of claim 28, wherein the complementary RNA and DNA molecules are synthetic nucleotide sequences.
- 30. The composition of claim 23, wherein the substrate is a cell or an organism.
- 31. The composition of claim 23, further comprising a carrier molecule, which carrier molecule is capable of being taken up by a cell.
- 32. A method for inhibiting the expression of a targeted gene in a substrate that expresses the targeted gene, comprising the steps of
a) providing a composition comprising a DNA-RNA hybrid capable of inhibiting the expression of said targeted gene in said substrate; and b) contacting said substrate with said composition under conditions such that the expression of said gene in said substrate is inhibited.
- 33. The method of claim 32, wherein said composition is the composition of claim 24 or claim 28 or both.
- 34. The method of claim 32, wherein said substrate expresses said targeted gene in vivo.
- 35. The method of claim 32, wherein said targeted gene comprises a gene selected from the group consisting of pathogenic nucleic acids, viral genes, mutated genes, oncogenes and unknown functional genes.
- 36. The method of claim 32, wherein said DNA-RNA hybrid inhibits β-catenin oncogene expression.
- 37. The method of claim 32, wherein said DNA-RNA hybrid inhibits bcl-2 drug-resistant gene expression.
- 38. The method of claim 32, wherein said substrate is a prokaryote.
- 39. The method of claim 38, wherein said prokaryote is a virus.
- 40. The method of claim 38, wherein said prokaryote is a bacterial cell.
- 41. The method of claim 32, wherein said substrate is an eukaryote or the cell of said eukaryote.
- 42. The method of claim 41, wherein said eukaryote is a vertebrate.
- 43. The method of claim 41, wherein said eukaryote is a mouse or rat.
- 44. The method of claim 41, wherein said eukaryote is a chimpanzee.
- 45. The method of claim 41, wherein said eukaryote is a human being.
- 46. An isolated nucleic acid molecule comprising a first strand of deoxynucleic acid (DNA) coupled to a second strand of riboxynucleic acid (RNA), wherein the RNA comprises nucleic acid sequence that is either homologous to or complementary to a messenger RNA (mRNA) molecule.
- 47. The isolated nucleic acid molecule of claim 46 comprises no nucleotide analog.
- 48. The isolated nucleic acid molecule of claim 46 comprises at least one nucleotide analog which is selected from the group consisting of inosine, xanthine, hypoxanthine, deoxyuracil, ribonucleotide in a DNA linkage, deoxyribonucleotide in an RNA linkage, 7-deaza-dNTP, labeled nucleotides, and their derivative analogs.
- 49. The isolated nucleic acid molecule of claim 48 wherein the derivative of said nucleotide analog is preferably selected from the group consisting of hexose-containing, 2′-5′ linked, phosphothio-linked, methylthio-linked, morpholino-linked and peptide-linked nucleotide analogs.
- 50. The isolated nucleic acid molecule of claim 48 wherein the nucleotide analog is a depurinated nucleotide.
- 51. The isolated nucleic acid molecule of claim 46, wherein the DNA and RNA are at least 20 percent complementary.
- 52. The isolated nucleic acid molecule of claim 51, wherein the DNA and RNA are about 95 percent complementary when the RNA comprises at least one palindromic sequence.
- 53. The isolated nucleic acid molecule of claim 52, wherein the RNA is at least about 45 percent complementary to the targeted mRNA.
- 54. The isolated nucleic acid molecule of claim 46, wherein the DNA comprises SEQ ID NO: 2.
- 55. The isolated nucleic acid molecule of claim 46 wherein the DNA comprises SEQ ID NO: 3.
- 56. The isolated nucleic acid molecule of claim 46 wherein the DNA comprises SEQ ID NO: 4.
- 57. The isolated nucleic acid molecule of claim 46 wherein the mRNA is expressed from a gene of interest.
- 58. The isolated nucleic acid molecule of claim 57 wherein the gene of interest is an oncogene.
- 59. The isolated nucleic acid molecule of claim 58, wherein the oncogene is β-catenin.
- 60. The isolated nucleic acid molecule of claim 57, wherein the gene of interest is a viral gene or genome.
- 61. The isolated nucleic acid molecule of claim 60, wherein the viral genome is a DNA or RNA molecule containing partial or full of the viral genome.
- 62. The isolated nucleic acid molecule of claim 61, wherein the viral genome is a plurality of viral genes from the HIV-1 genome ranging from about +1890 to +2230 bases.
- 63. The isolated nucleic acid molecule of claim 46, wherein the gene of interest expresses a protein.
- 64. The isolated nucleic acid molecule of claim 63, wherein the protein is tyrosinase.
- 65. The isolated nucleic acid molecule of claim 52, wherein the nucleic acid sequence of the RNA is at least about 48% complementary to the corresponding portion of the messenger riboxynucleic acid molecule.
- 66. The isolated nucleic acid molecule of claim 23 or claim 46, wherein the nucleic acid molecule is double stranded nucleotide sequences ranging from about 20 to about 10,000 basepairs.
- 67. The isolated nucleic acid molecule of claim 23 or claim 46, wherein the double stranded nucleic acid molecule is sized ranging from about 20 to about 150 basepairs.
- 68. The isolated nucleic acid molecule of claim 23 or claim 46, wherein the DNA comprises at least one labeled deoxyribonucleotide.
- 69. The isolated nucleic acid molecule of claim 23 or claim 46, wherein the labeled ribonucleotide is labeled with a molecule selected from the group consisting of a fluorophore, a hapten, a ligand, an enzyme, and a radioactive molecule.
- 70. The use of the isolated nucleic acid molecule of claim 23 or claim 46 to alter the characteristic of an eukaryotic cell.
- 71. The use of claim 23 or claim 46 wherein the characteristic is selected from the group consisting of (a) expression of a protein; (b) cell division rate; (c) pigmentation.
- 72. The use of claim 23 or claim 46 wherein the isolate nucleic acid molecule has an effect that lasts at least three days.
- 73. The use of the isolated nucleic acid molecule of claim 23 or claim 46 to inhibit the expression of messenger RNA in a cell.
- 74. The use of claim 23 or claim 46 wherein the messenger RNA is transcribed from a gene selected from a group consisting of viral gene, oncogene, enzyme.
- 75. The use of claim 23 or claim 46 wherein the isolated nucleic acid molecule is used at a concentration ranging from about 1 nM to about 750 nM.
- 76. The use of claim 23 or claim 46 wherein the isolated nucleic acid molecule, wherein the concentration ranges from about 5 nM to about 50 nM.
- 77. A composition comprising the isolated nucleic acid molecule of claim 23 or claim 46 and a transfection agent.
- 78. The composition of claim 23 or claim 46 wherein the transfection agent is selected from the group consisting of saline solution, calcium phosphate, liposomes, lipid derivatives, dextran sulfate, and polymers.
- 79. A composition comprising multiple species of the isolated nucleic acid molecule of claim 23 or claim 46, wherein each species has a different nucleic acid sequence than another.
- 80. The composition of claim 23 or claim 46 wherein the number of species ranges from two to ten.
- 81. An article of manufacture comprising a container comprising the isolated nucleic acid of claim 23 or claim 46 and a label providing information on the use of the isolated nucleic acid of claim 23 or claim 46.
- 82. The article of manufacture of claim 23 or claim 46 wherein the label is affixed to the container.
- 83. The article of manufacture of claim 23 or claim 46 wherein the label is an instruction sheet or an instruction manual.
- 84. A method of making a DNA-RNA hybrid molecule capable of altering the characteristic of an eukaryotic cell, the method comprising the steps of:
a) synthesizing an RNA molecule with a sequence either homologous or complementary to a RNA species in a cell; b) synthesizing a DNA molecule with a sequence complementary to the RNA molecule of (a); c) forming a DNA-RNA hybrid molecule from the RNA molecule of (a) and the DNA molecule of (b); wherein the DNA-RNA hybrid molecule is capable of altering the characteristic of the cell.
- 85. The method of claim 84 wherein the hybrid comprises at least one nucleotide analog.
- 86. The method of claim 85 wherein the at least one nucleotide analog is selected from the group consisting of inosine, xanthine, hypoxanthine, deoxyuracil, ribonucleotide in a DNA linkage, deoxyribonucleotide in an RNA linkage, 7-deaza-dNTP, labeled nucleotides, and their derivatives which are preferably selected from the group consisting of hexose-containing, 2′-5′ linked, phosphothio-linked, methylthio-linked, morpholino-linked and peptide-linked nucleotide analogs.
- 87. The method of claim 84 further comprising the step of treating the DNA molecule with deaminase.
- 88. The method of claim 84, wherein the DNA and RNA are at least 45% complementary.
- 89. The method of claim 84 wherein the step of synthesizing DNA molecule is by chemical synthesis.
- 90. The method of claim 84 wherein the step of synthesizing the DNA molecule is by reverse transcription from an RNA molecule.
- 91. The method of claim 84 wherein the step of synthesizing the DNA molecule is by polymerase chain reaction from the a DNA molecule.
- 92. The method of claim 84 wherein the DNA molecule is a viral genome.
- 93. The method of claim 84 wherein the step of synthesizing the RNA molecule is by chemical synthesis.
- 94. The method of claim 84 wherein the step of synthesizing the RNA molecule is by in vitro transcription or viral replication.
- 95. The method of claim 84 wherein the RNA molecule is a viral genome.
- 96. The method of claim 84 wherein the DNA-RNA hybrid is formed by repeated steps of (1) in vitro transcription from a double-stranded DNA template molecule and (2) reverse transcription of the RNA molecule product of (1)
- 97. The method of claim 96 wherein the repeated steps are repeated at least once.
- 98. The method of claim 96 wherein the double-stranded DNA molecule is a cDNA molecule generated from the reverse transcription and polymerase chain reaction of a mRNA molecule using a primer comprising nucleic acid sequence for the RNA polymerase promoter.
- 99. The method of claim 96 wherein the double-stranded DNA is generated by hybridization of chemically synthesized DNA sequences.
- 100. The method of claim 96 wherein the double-stranded DNA is a plasmid or viral vector.
- 101. The method of altering a characteristic of a eukaryotic cell, the method comprising introducing into the eukaryotic cell a nucleic acid molecule mixture comprising a first strand DNA molecule and a second strand RNA molecule, wherein the RNA molecule is complementary to the DNA.
- 102. The method of altering a characteristic of a eukaryotic cell, wherein either the RNA or the DNA molecule is complementary to a messenger RNA species in the cell.
- 103. The method of claims 89 or 92, wherein the synthesized DNA molecule contains none or at least one ribonucleotide or its analog.
- 104. The method of claims 93 or 94, wherein the synthesized RNA molecule contains none or at least one deoxyribonucleotide or its analog.
- 105. The method of claim 104, wherein the synthesized RNA molecule containing at least one deoxyribonucleotide analog is to increase RNAi phenomenon induction and to reduce interferon-related non-specific effects.
CROSS REFERENCE OF RELATED APPLICATION
[0001] This is a Continuation-In-Part application claiming priority to a non-provisional application, application Ser. No. 10/052,486, filed on Jan. 22, 2002, entitled GENE SILENCING USING SENSE DNA AND ANTISENSE RNA HYBRID CONSTRUCTS, and a provisional application, application No. 60/351,183, filed on Nov. 12, 2001, entitled GENE SILENCING USING SENSE DNA AND ANTISENSE RNA HYBRID CONSTRUCTS, which is hereby incorporated by reference as if fully set forth herein.
Provisional Applications (1)
|
Number |
Date |
Country |
|
60351183 |
Nov 2001 |
US |
Continuation in Parts (1)
|
Number |
Date |
Country |
Parent |
10052486 |
Jan 2002 |
US |
Child |
10393450 |
Mar 2003 |
US |