Claims
- 1. A method for modifying a nucleotide sequence in the genome of a cell, comprising:
providing a cell in S-phase of the cell cycle; and contacting the cell with a DNA modifying molecule that modifies the nucleotide sequence.
- 2. The method of claim 1, wherein contacting the cell comprises activating the DNA modifying molecule to modify the nucleotide sequence.
- 3. The method of claim 2, wherein the DNA modifying molecule is activatable by radiation, and activating the DNA modifying molecule comprises irradiating the molecule with radiation.
- 4. The method of claim 3, wherein the radiation is ultraviolet radiation.
- 5. The method of claim 1, wherein the cell is contacted with the DNA modifying molecule at a cell cycle phase wherein DNA modification by the DNA modifying molecule occurs at a higher frequency relative to a second cell cycle phase.
- 6. The method of claim 1, wherein the cell is contacted with the DNA modifying molecule in mid to late S phase.
- 7. The method of claim 1, wherein providing a cell comprises providing a population of cells.
- 8. The method of claim 7, further comprising synchronizing the population of cells to yield a synchronized cell population, and contacting the cell comprises contacting the synchronized cell population with the DNA modifying molecule.
- 9. The method according to claim 8, wherein at least 50% of the synchronized cell population is synchronized in S phase when the synchronized cell population is contacted with the DNA modifying molecule.
- 10. The method according to claim 9, wherein at least 75% of the synchronized cell population is synchronized in S phase when the synchronized cell population is contacted with the DNA modifying molecule.
- 11. The method of claim 1, wherein the cell is a human cell.
- 12. The method of claim 1, wherein the cell is a fertilized egg cell from an animal selected from the group consisting of a mouse, hamster, sheep, pig, rabbit, or cow.
- 13. The method of claim 1, wherein the cell is a mouse cell selected from the group consisting of a blastomere cell, an eight-cell embryo cell, a blastocoele cell, a midgestation cell embryo cell, or an embryonic stem cell.
- 14. The method of claim 1, wherein the DNA modifying molecule comprises a DNA targeting agent selected from the group consisting of peptide nucleic acids, polyamides, triplex forming oligonucleotides, zinc finger proteins and combinations thereof.
- 15. The method of claim 1, wherein the DNA modifying molecule comprises a modified oligonucleotide.
- 16. The method of claim 15, wherein the modified oligonucleotide is a triplex forming oligonucleotide.
- 17. The method of claim 15, wherein the modified oligonucleotide comprises at least one 2′-O-alkylated residue.
- 18. The method of claim 17, wherein the 2′-O-alkylated residue is a 2′-aminoalkoxy residue.
- 19. The method of claim 17, wherein the at least one 2′-O-alkylated residue is a pyrimidine residue.
- 20. The method of claim 15, wherein the modified oligonucleotide comprises from 10 to 25 residues.
- 21. The method of claim 20, wherein the modified oligonucleotide comprises no more than four 2′-O-alkylated residues.
- 22. The method of claim 21, wherein the modified oligonucleotide comprises multiple contiguous 2′-aminoalkoxy residues.
- 23. The method of claim 21, wherein the modified oligonucleotide comprises four contiguous 2′-aminoalkoxy residues.
- 24. The method of claim 15, wherein the modified oligonucleotide comprises at least one unit according to formula I
- 25. A cell modified by the method of claim 1.
- 26. A modified oligonucleotide comprising at least one unit according to formula I
- 27. The oligonucleotide of claim 26, wherein A is a residue of xanthine, hypoxanthine, adenine, 2-aminoadenine, guanine, cytosine, thymine, 6-thioguanine, uracil, 5-methylcytosine, 5-propynyluracil, 5-fluorouracil, 5-propynylcytosine, 2,6-diaminopurine, purine, 7-deazaadenine, 7-deazaguanine, 5-propynyluracil, isoguanine, 2-aminopurine, 6-methyluracil, 4-thiouracil, 2-pyrimidone, N,N-dimethylguanine, bromouracil, aminopyridine, or a functional equivalent thereof.
- 28. The oligonucleotide of claim 26, wherein A is a pyrimidine residue.
- 29. The oligonucleotide of claim 26, wherein A is a residue of cytosine or 5-substituted cytosine.
- 30. The oligonucleotide of claim 26, wherein A is a residue of uridine or 5-substituted uridine.
- 31. The oligonucleotide of claim 26, wherein X and Y together form a phosphodiester, a phosphorothioate, methylphosphonate, H-phosphonate, or an amide bond between adjacent nucleosides or nucleoside analogs or together form an analog of a phosphodiester bond.
- 32. The oligonucleotide of claim 26, wherein Z is a lower alkyl group.
- 33. The oligonucleotide of claim 26, wherein Z is —CH2CH2—.
- 34. The oligonucleotide of claim 26, wherein R1 and R2 are, independently selected from the group consisting of hydrogen, methyl, ethyl, propyl, or phenyl.
- 35. The oligonucleotide of claim 26, wherein V and W are oxygen.
- 36. The oligonucleotide of claim 26, wherein A is a residue of 5-methylcytosine or thymine; V and W are oxygen; X and Y together form a phosphodiester bond; Z is —CH2CH2—; and R1 and R2 are hydrogen.
- 37. The oligonucleotide of claim 26, wherein the mutagen comprises a moiety selected from the group consisting of radionuclides, crosslinkers, alkylators, base modifiers, DNA breakers, free radical generators and combinations thereof.
- 38. The oligonucleotide of claim 26, wherein the mutagen comprises a moiety selected from the group consisting of bleomycin, cyclopropapyrroloindoles, phenanthodihydrodioxins, indolocarbazoles, napthalene diimide, chlorambucil, mitomycin derivatives, enediyne derivatives, hematoporphyrin derivatives, coumarin derivatives, oxazolopyridocarbazole, daunomycine, anthraquinone, acridine orange, cis platinum derivatives, radionuclides, boron agents, and combinations thereof.
- 39. The oligonucleotide of claim 26, wherein the mutagen comprises an intercalator.
- 40. The oligonucleotide of claim 26, wherein the mutagen is activatable by irradiation.
- 41. The oligonucleotide of claim 26, wherein the mutagen comprises a psoralen derivative.
- 42. The oligonucleotide of claim 26, wherein the oligonucleotide contains from 10 to 25 nucleotides.
- 43. The oligonucleotide of claim 26, wherein the oligonucleotide comprises multiple units having formula I.
- 44. The oligonucleotide of claim 43, wherein two or more of the multiple units having formula I are localized to a region of the oligonucleotide that is less than the entire length of the oligonucleotide.
- 45. The oligonucleotide of claim 43, wherein two or more of the multiple units having formula I are contiguous.
- 46. The oligonucleotide of claim 44, wherein the region of the oligonucleotide is less than 6 residues in length.
- 47. The oligonucleotide of claim 23, comprising at least 3 but no more than 4 units having formula I.
- 48. The oligonucleotide of claim 47, wherein the units having formula I are contiguous.
- 49. A pharmaceutical composition comprising the oligonucleotide of claim 26 and a pharmaceutically acceptable carrier.
- 50. A method for mutating a nucleotide sequence in the genome of a cell, comprising contacting the cell with a modified oligonucleotide to produce a mutation of the nucleotide sequence, wherein the modified oligonucleotide comprises at least one unit according to formula I, or a salt thereof.
- 51. The method of claim 50, wherein mutating the nucleotide sequence comprises introducing a deletion, insertion, substitution, strand break, adduct formation, gene conversion, or recombination of a novel sequence.
- 52. The method of claim 50, wherein the cell is human.
- 53. The method of claim 50, wherein the cell is non-human.
- 54. The method of claim 50, wherein the cell is a fertilized egg cell from an animal selected from the group consisting of a mouse, hamster, sheep, pig, rabbit, or cow.
- 55. The method of claim 50, wherein the cell is a mouse cell selected from the group consisting of a blastomere cell, an eight-cell embryo cell, a blastocoele cell, a midgestation cell embryo cell, or an embryonic stem cell.
- 56. The method of claim 50, wherein contacting the cell with the modified oligonucleotide comprises contacting a population of cells.
- 57. The method of claim 56, wherein contacting the population of cells with the modified oligonucleotide comprises synchronizing the population of cells to yield a synchronized cell population.
- 58. The method of claim 57, wherein at least 50% of the synchronized cell population is synchronized in S phase when the synchronized cell population is contacted with the modified oligonucleotide.
- 59. The method of claim 57, wherein at least 75% of the synchronized cell population is synchronized in S phase when the synchronized cell population is contacted with the modified oligonucleotide.
- 60. The method of claim 50, wherein contacting the cell with the modified oligonucleotide comprises contacting the cell with the modified nucleotide when the cell is in mid to late S phase.
- 61. A cell produced by the method of claim 50.
- 62. The cell of claim 61, wherein the cell is incorporated in an animal.
- 63. The cell of claim 62, wherein the animal is a patient and incorporation of the cell ameliorates a medical condition.
- 64. A cell comprising the modified oligonucleotide of claim 26.
- 65. A vector comprising the modified oligonucleotide of claim 26.
- 66. The vector of claim 65, wherein the vector is a nucleic acid vector.
- 67. The vector of claim 65, wherein the vector is a viral vector.
- 68. A modified oligonucleotide, comprising:
a first unit according to formula I; at least a second unit according to formula I, wherein X or Y comprise a mutagenic group, and V, W, Z, R1, R2, R3, R4, and R5 are selected as above; or a salt thereof.
- 69. The modified oligonucleotide according to claim 68, wherein the mutagenic group of the second unit comprises an intercalator.
- 70. The modified oligonucleotide according to claim 68, wherein the mutagenic group of the second unit comprises a group according to the formula
- 71. A modified oligonucleotide, comprising no more than four units according to formula I, or a salt thereof.
- 72. The oligonucleotide according to claim 71, wherein the oligonucleotide is from 10 to 25 nucleic acid residues in length.
- 73. The oligonucleotide according to claim 71, wherein the oligonucleotide includes at least three but no more than four units according to formula I.
- 74. The oligonucleotide according to claim 71, further comprising a mutagen.
- 75. The oligonucleotide according to claim 74, wherein the mutagen comprises an intercalator.
- 76. The oligonucleotide according to claim 74, wherein the mutagen comprises a psoralen derivative.
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of U.S. Provisional Application No. 60/378,025, filed May 13, 2002, which is incorporated herein by reference.
Provisional Applications (1)
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Number |
Date |
Country |
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60378025 |
May 2002 |
US |