Thio-siRNA aptamers

• US 20060172925A1
• Filed: 10/19/2004
• Published: 08/03/2006
• Est. Priority Date: 10/26/1998
• Status: Abandoned Application

• Current Assignee: Board of Regents of the University of Texas System (University of Texas System)
• Original Assignee: Board of Regents of the University of Texas System (University of Texas System)
• Inventors: Gorenstein, David G., Yang, Xianbin
• Application Number:
  US10/968,574
• Publication Number:
  US 20060172925A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 15/1131 against viruses
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2310/315 Phosphorothioates
• CPC Class Codes: C12N 2330/31 Libraries, arrays

Abstract

The present invention includes thioaptamers that are partially thio-modified, methods and compositions for the isolation, selection, improvement, characterization and use of RNA and DNA thioaptamers for gene silencing, including degradative and non-degradative interference with translation.

Claims

1. An isolated thioaptamer that mediates gene silencing, wherein the thioaptamer comprises a double-stranded hybrid thioaptamer.

2. The thioaptamer of claim 1, wherein the double-stranded hybrid thioaptamer is selected from RNA:

DNA, RNA;

RNA, DNA;

DNA, PNA;

DNA or PNA;

RNA.

3. The thioaptamer of claim 1, wherein the double-stranded hybrid thioaptamer is thioated at one or more locations.

4. The thioaptamer of claim 1, wherein the double-stranded hybrid thioaptamer is dithioated.

5. The thioaptamer of claim 1, further comprising a terminal 3′

hydroxyl group.

6. The thioaptamer of claim 1, wherein the double-stranded hybrid thioaptamer comprises a perfect complementarity match to a target gene and gene silencing occurs by mRNA cleavage.

7. The thioaptamer of claim 1, further comprising a double-stranded hybrid thioaptamer having an imperfect complementarity match to a target gene and gene silencing occurs by mRNA cleavage.

8. The thioaptamer of claim 1, further comprising a double-stranded hybrid thioaptamer having an perfect complementarity match to a target gene and gene silencing occurs by repressed translation of mRNA to protein.

9. The thioaptamer of claim 1, wherein the thioaptamer comprises a double-stranded hybrid thioaptamer with an imperfect complementarity match to a target gene and gene silencing occurs by repressed translation of mRNA to protein.

10. The thioaptamer of claim 1, wherein one strand of the double-stranded hybrid thioaptamer comprises one or more of the following:

rATP(α

S), rUTP(α

S), rGTP(α

S), rCTP(α

S), rATP(α

S₂), rUTP(α

S₂), rGTP(α

S₂) or rCTP(α

S₂).

11. The thioaptamer of claim 1, wherein one strand of the double-stranded hybrid thioaptamer comprises one or more of the following:

dATP(α

S), dTTP(α

S), dGTP(α

S), dCTP(α

S), dATP(α

S₂), dTTP(α

S₂), dGTP(α

S₂), or dTTP(α

S₂).

12. An isolated thioaptamer selected from the group consisting of SEQ ID NO.:

26 to SEQ ID NO.;

57.

13. An isolated thioaptamer that mediates gene silencing, wherein the thioaptamer comprises a combination of short interfering DNA (siDNA);

a micro, interfering DNA (miDNA);

a small, temporal DNA (stDNA);

a short, hairpin DNA (shDNA);

short interfering RNA (siRNA);

a micro, interfering RNA (mRNA);

a small, temporal RNA (stRNA);

or a short, hairpin RNA (shRNA).

14. The thioaptamer of claim 13, wherein the thioaptamer is monothioated.

15. The thioaptamer of claim 13, wherein the thioaptamer is thioated at one or more locations.

16. The thioaptamer of claim 13, further comprising a terminal hydroxyl group.

17. The thioaptamer of claim 13, wherein the thioaptamer comprises one or more of the following:

dATP(α

S), dTTP(α

S), dGTP(α

S), dCTP(α

S), dATP(α

S₂), dTTP(α

S₂), dGTP(α

S₂), dTTP(α

S₂), rATP(α

S), rUTP(α

S), rGTP(α

S), rCTP(α

S), rATP(α

S₂), rUTP(α

S₂), rGTP(α

S₂) or rCTP(α

S₂).

18. A method of mediating gene silencing of a target gene in a cell or organism comprising the steps of:

introducing a double-stranded hybrid thioaptamer 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.

19. The method of claim 18, wherein the double-stranded hybrid thioaptamer is selected from RNA:

DNA, RNA;

RNA, DNA;

DNA, PNA;

DNA or PNA;

RNA.

20. The method of claim 18, wherein the target gene is selected from a viral gene, a cellular gene, a fungal gene, an extrachromosomal gene, a chromosomal gene, a genomic gene, a messenger RNA, or a RNAi.

21. The method of claim 18, wherein gene silencing is defined further as degradation of an mRNA transcript of the target gene that is cleaved in the presence of the thioaptamer before it can express a protein.

22. The method of claim 18, wherein gene silencing is defined further as regulation of translation of the target gene when the thioaptamer binds an mRNA transcript of the target gene at or about its 3′

UTR.

23. A method of examining the function of a gene in a cell or organism comprising the steps of:

introducing a double-stranded hybrid thioaptamer 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.

24. The method of claim 23, wherein the double-stranded hybrid thioaptamer is selected from RNA:

DNA, RNA;

RNA, DNA;

DNA, PNA;

DNA or PNA;

RNA.

25. A method of assessing whether a gene product is a suitable target for drug discovery comprising the steps of:

introducing an double-stranded hybrid thioaptamer that mediates gene silencing 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.

26. The method of claim 25, wherein the double-stranded hybrid thioaptamer is selected from RNA:

DNA, RNA;

RNA, DNA;

DNA, PNA;

DNA or PNA;

RNA.

27. A pharmaceutical composition comprising a double-stranded hybrid thioaptamer that mediates thioaptamer gene silencing and an appropriate carrier.

28. A method for reducing the expression of a gene in a cell, comprising the steps of:

selecting a double-stranded hybrid 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.

29. The method of claim 28, wherein the double-stranded hybrid thioaptamer is selected from RNA:

DNA, RNA;

RNA, DNA;

DNA, PNA;

DNA or PNA;

RNA.

30. The method of claim 28, wherein the thioaptamer comprises double-stranded hybrid thioaptamer with a perfect complementarity match to a target gene and gene silencing occurs by mRNA cleavage.

31. The method of claim 28, wherein the thioaptamer comprises double-stranded hybrid thioaptamer with a imperfect complementarity match to a target gene and gene silencing occurs by mRNA cleavage.

32. A method for attenuating expression of a target gene in cultured cells, comprising the step of:

introducing a double-stranded hybrid thioaptamer into the cells in an amount sufficient to attenuate expression of the target gene, wherein the double-stranded hybrid 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.

33. The method of claim 32, wherein the cell is in cell culture, a virus, a mammalian cell, a human cell or a stem cell.

34. A method of producing a double-stranded hybrid thioaptamer comprising the steps of:

combining a double-stranded hybrid thioaptamer precursor 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 hybrid thioaptamer is processed to the mature thioaptamer.

35. The method of claim 34, wherein the double-stranded hybrid thioaptamer is selected from RNA:

DNA, RNA;

RNA, DNA;

DNA, PNA;

DNA or PNA;

RNA.

36. The method of claim 34, further comprising isolating the double-stranded hybrid thioaptamer from the precursor-extract mixture.

37. The method of claim 34, further comprising the steps of:

determining the sequence of the double-stranded hybrid thioaptamer and the location of one or more thio-modifications to the thioaptamer; and

chemically synthesizing the thioaptamer.

38. A thioaptamer produced by the method of claim 34.

39. A method of mediating gene silencing of a target gene in a cell or organism comprising the steps of:

introducing a thioaptamer into the cell or organism, wherein the thioaptamer comprises a combination of short interfering DNA (siDNA);

a micro, interfering DNA (miDNA);

a small, temporal DNA (stDNA);

a short, hairpin DNA (shDNA);

short interfering RNA (siRNA);

a micro, interfering RNA (mRNA);

a small, temporal RNA (stRNA);

or a short, hairpin RNA (shRNA); 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.

40. The method of claim 39, wherein the cell is in cell culture, a virus, a mammalian cell, a human cell or a stem cell.

41. The method of claim 39, wherein gene silencing is defined further as degradation of an mRNA transcript of the target gene that is cleaved in the presence of the thioaptamer before it can express a protein.

42. The method of claim 39, wherein gene silencing is defined further as regulation of translation of the target gene when the thioaptamer binds an mRNA transcript of the target gene at or about its 3′

UTR.

43. The thioaptamer of claim 39, wherein the thioaptamer comprises a combination of short interfering DNA (siDNA);

a micro, interfering DNA (miDNA);

a small, temporal DNA (stDNA);

a short, hairpin DNA (shDNA);

short interfering RNA (siRNA);

a micro, interfering RNA (mRNA);

a small, temporal RNA (stRNA);

or a short, hairpin RNA (shRNA) with a perfect complementarity match to a target gene and gene silencing occurs by mRNA cleavage.

44. The thioaptamer of claim 39, wherein the thioaptamer comprises a combination of short interfering DNA (siDNA);

a micro, interfering DNA (miDNA);

a small, temporal DNA (stDNA);

a short, hairpin DNA (shDNA);

short interfering RNA (siRNA);

a micro, interfering RNA (mRNA);

a small, temporal RNA (stRNA);

or a short, hairpin RNA (shRNA) with an imperfect complementarity match to a target gene and gene silencing occurs by repressed translation of mRNA to protein.

45. A method of examining the function of a gene in a cell or organism comprising the steps of:

introducing a thioaptamer comprises a combination of short interfering DNA (siDNA);

a micro, interfering DNA (miDNA);

a small, temporal DNA (stDNA);

a short, hairpin DNA (shDNA);

short interfering RNA (siRNA);

a micro, interfering RNA (mRNA);

a small, temporal RNA (stRNA);

or a short, hairpin RNA (shRNA) 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.

46. A method of assessing whether a gene product is a suitable target for drug discovery comprising the steps of:

introducing a thioaptamer comprises a combination of short interfering DNA (siDNA);

a micro, interfering DNA (miDNA);

a small, temporal DNA (stDNA);

a short, hairpin DNA (shDNA);

short interfering RNA (siRNA);

a micro, interfering RNA (mRNA);

a small, temporal RNA (stRNA);

or a short, hairpin RNA (shRNA) that mediates gene silencing 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.

47. A pharmaceutical composition comprising a thioaptamer that mediates thioaptamer gene silencing and an appropriate carrier, wherein the thioaptamer comprises a combination of short interfering DNA (siDNA);

a micro, interfering DNA (miDNA);

a small, temporal DNA (stDNA);

a short, hairpin DNA (shDNA);

short interfering RNA (siRNA);

a micro, interfering RNA (mRNA);

a small, temporal RNA (stRNA);

or a short, hairpin RNA (shRNA).

48. A method for reducing the expression of a gene in a cell, comprising the steps of:

selecting a thioaptamer comprises a combination of short interfering DNA (siDNA);

a micro, interfering DNA (miDNA);

a small, temporal DNA (stDNA);

a short, hairpin DNA (shDNA);

short interfering RNA (siRNA);

a micro, interfering RNA (mRNA);

a small, temporal RNA (stRNA);

or a short, hairpin RNA (shRNA) 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.

49. The method of claim 48, wherein the thioaptamer comprises an imperfect complementarity match to a target gene and gene silencing occurs by mRNA cleavage.

50. The method of claim 48, wherein the thioaptamer comprises a perfect complementarity match to a target gene and gene silencing occurs by mRNA cleavage.

51. A method for attenuating expression of a target gene in cultured cells, comprising the step of:

introducing a thioaptamer comprises a combination of short interfering DNA (siDNA);

a micro, interfering DNA (miDNA);

a small, temporal DNA (stDNA);

a short, hairpin DNA (shDNA);

short interfering RNA (siRNA);

a micro, interfering RNA (mRNA);

a small, temporal RNA (stRNA);

or a short, hairpin RNA (shRNA) into the cells in an amount sufficient to attenuate expression of the target gene, wherein the 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.

52. The method of claim 51, wherein the cell is in cell culture, culture, a virus, a mammalian cell, a human cell or a stem cell.

53. A method of producing a thioaptamer comprising the steps of:

combining a combination of short interfering DNA (siDNA);

a micro, interfering DNA (miDNA);

a small, temporal DNA (stDNA);

a short, hairpin DNA (shDNA);

short interfering RNA (siRNA);

a micro, interfering RNA (mRNA);

a small, temporal RNA (stRNA);

or a short, hairpin RNA (shRNA) thioaptamer precursor with a soluble extract that mediates gene silencing, thereby producing a precursor-extract mixture; and

maintaining the precursor-extract mixture under conditions in which a combination of short interfering DNA (siDNA);

a micro, interfering DNA (miDNA);

a small, temporal DNA (stDNA);

a short, hairpin DNA (shDNA);

short interfering RNA (siRNA);

a micro, interfering RNA (mRNA);

a small, temporal RNA (stRNA);

or a short, hairpin RNA (shRNA) thioaptamer is processed to the mature thioaptamer.

54. The method of claim 53, further comprising isolating the thioaptamer from the precursor-extract mixture.

55. The method of claim 53, further comprising the step of determining the sequence of the thioaptamer and the location of one or more thio-modifications to the mature thioaptamer.

56. A thioaptamer produced by the method of claim 53.

57. The thioaptamer of claim 56, comprising SEQ ID NO.:

26 to SEQ ID NO.;

57.