Thio-siRNA aptamers
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Abstract
The present invention includes thioaptamers, methods and libraries for the isolation, selection, improvement, characterization and use of RNA thioaptamers for gene silencing, including degradative and non-degradative interference with translation.
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Citations
95 Claims
- 1. An isolated thioaptamer that mediates gene silencing.
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18. A method of producing a mature thioaptamer of from about 21 to about 23 nucleotides in length comprising the steps of:
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combining a double-stranded precursor thioaptamer with a soluble extract that mediates gene silencing, thereby producing a precursor-extract mixture; and
maintaining the precursor-extract mixture under conditions in which the double-stranded thioaptamer is processed to the mature thioaptamer of from about 21 to about 23 nucleotides in length. - View Dependent Claims (19, 20, 21, 22)
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23. A method of mediating gene silencing of a target gene in a cell or organism comprising the steps of:
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introducing a thioaptamer of from about 21 to about 23 nucleotides in length into the cell or organism; and
maintaining the cell or organism under conditions in which gene silencing occurs, thereby mediating expression of the target gene in the cell or organism. - View Dependent Claims (24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34)
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35. A method of examining the function of a gene in a cell or organism comprising the steps of:
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introducing a thioaptamer of from about 21 to about 23 nucleotides that targets an mRNA of the gene for gene silencing into the cell or organism, thereby producing a test cell or test organism;
maintaining the test cell or test organism under conditions under which gene silencing of mRNA of the gene occurs, thereby producing a test cell or test organism in which mRNA of the gene is silenced; and
observing the phenotype of the test cell or test organism against an appropriate control cell or control organism to provide information about the function of the gene.
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36. A method of assessing whether a gene product is a suitable target for drug discovery comprising the steps of:
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introducing an RNA thioaptamer that mediates gene silencing of from about 21 to about 25 nucleotides into a cell or organism under conditions in which gene silencing of an mRNA for the target gene results in decreased expression of the gene; and
determining the effect of the decreased expression of the gene on the cell or organism, wherein if decreased expression has an effect, then the gene product is a target for drug discovery.
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37. A pharmaceutical composition comprising a thioaptamer of from about 21 to about 25 nucleotides that mediates thioaptamer gene silencing and an appropriate carrier.
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38. A method of identifying target sites within an mRNA that are efficiently targeted for gene silencing, comprising the step of:
combining an RNA thioaptamer corresponding to a sequence of a labeled mRNA to be degraded under conditions in which labeled mRNA is degraded. - View Dependent Claims (39, 40, 41)
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42. A method of identifying target sites within an mRNA that are efficiently targeted for gene silencing, comprising the step of:
combining an RNA thioaptamer corresponding to a sequence of a labeled mRNA under conditions in which labeled mRNA is not degraded and the protein level is reduced. - View Dependent Claims (43, 44)
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45. A combinatorial thioaptamer library comprising:
two or more unique thioaptamers that comprise a combination of backbone modifications and sequence that mediates gene silencing of an mRNA to which it corresponds. - View Dependent Claims (46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60)
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61. A one-bead, one-thioaptamer combinatorial library comprising:
two or more beads, wherein attached to each bead is a unique thioaptamer comprising a single unique sequence, wherein each unique thioaptamer comprises a unique mix of modified and unmodified nucleotides and wherein the thioaptamer mediates gene silencing of an mRNA to which it corresponds.
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62. A one-bead, one-thioaptamer combinatorial library comprising:
two or more beads, wherein attached to each bead is a unique thioaptamer comprising an imperfect complementarity match to a target gene to form a thioaptamer-bead, wherein each unique thioaptamer-bead comprises a unique mix of modified and unmodified nucleotides and wherein the thioaptamer mediates gene silencing of an mRNA to which it has imperfect complementarity.
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62-1. A combinatorial library comprising:
a bead library of thioaptamer libraries, wherein each bead comprises a thioaptamer library of imperfect complementarity to a target sequence for gene silencing.
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63. A method for reducing the expression of a gene in a cell, comprising the steps of:
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selecting a thioaptamer that mediates gene silencing of the gene to which it corresponds; and
introducing the thioaptamer into the cell, wherein the thioaptamer mediates RNA interference of a targeted sequence. - View Dependent Claims (64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82)
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83. A method for attenuating expression of a target gene in cultured cells, comprising the step of:
introducing an RNA thioaptamer into the cells in an amount sufficient to attenuate expression of the target gene, wherein the RNA thioaptamer comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the target gene and mediates attenuation of protein expression for a gene to which it corresponds. - View Dependent Claims (84, 85, 86, 87, 88, 89, 90, 91, 92)
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93. A method for attenuating expression of a target gene in a mammalian cell, comprising the steps of:
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introducing into the cell a thioaptamer in an amount sufficient to attenuate expression of the target gene, wherein the thioaptamer mediates gene silencing of a nucleic acid to which it hybridizes under stringent conditions; and
activating a gene silencing activity in the cell. - View Dependent Claims (94, 95)
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Specification