Claims
- 1. A composition for modulating transcription of a gene, comprising:
a non-peptidic DNA binding domain, a transcriptional effector, one end of the linker being covalently bound to the DNA binding domain, and the other end of the linker being covalently bound to the transcriptional effector, and a flexible linker.
- 2. The composition of claim 1, wherein the transcriptional effector activates transcription.
- 3. The composition of claim 1, wherein the transcriptional effector represses transcription.
- 4. The composition of claim 1, wherein the transcriptional effector binds a co-effecting protein.
- 5. The composition of claim 3, wherein the transcriptional repressor binds a histone or histone-modifying protein.
- 6. The composition of claim 1, wherein the DNA binding domain comprises a nucleic acid.
- 7. The composition of claim 6, wherein the nucleic acid is a modified nucleic acid.
- 8. The composition of claim 7, wherein the modified nucleic acid is selected from a group of a nucleic acid having a modified backbone, a nucleic acid having a modified base, and combinations thereof.
- 9. The composition of claim 7, wherein the linker is bound to 3′ the end or the 5′ end of the nucleic acid.
- 10. The composition of claim 8, wherein the modified backbone is selected from the group consisting of phosphorothioates and peptide nucleic acids.
- 11. The composition of claim 1, wherein the DNA binding domain comprises a peptidic nucleic acid.
- 12. The composition of claim 1, wherein the DNA binding domain comprises a polyamide peptide analog.
- 13. The composition of claim 1, wherein the DNA binding domain comprises a DNA-binding natural product.
- 14. The composition of claim 1, wherein the DNA-binding domain comprises an antibiotic or moieties containing small organic material.
- 15. The composition of claim 1, wherein the DNA binding domain does not contain a plurality of pyrrole or imidazole groups.
- 16. The composition of claim 1, wherein the transcriptional effector is a polypeptide sequence.
- 17. The composition of claim 16, wherein the amino terminus of the polypeptide sequence is covalently bound to the linker.
- 18. The composition of claim 2, wherein the transcriptional activator is a polypeptide sequence, and the polypeptide sequence comprises at least one copy of an activator sequence of amino acids from herpes virus protein VP16 selected from the group consisting of comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:8 and SEQ ID NO:12.
- 19. The composition of claim 1, wherein the transcriptional effector is a non-peptidic organic moiety.
- 20. The composition of claim 1, wherein the transcriptional effector moiety has a molecular weight of less than about 3,000 daltons.
- 21. The composition of claim 1, wherein the transcriptional effector moiety has a molecular weight of less than about 1,500 daltons.
- 22. The composition of claim 1, wherein the flexible linker comprises a polyglycol.
- 23. The composition of claim 22, wherein the flexible linker polyglycol contains at least six glycol units.
- 24. The composition of claim 1, wherein the flexible linker comprises one or more units selected from the group consisting of nucleotides, peptides, lower alkyls and oxygen-containing alkyl groups.
- 25. The composition of claim 1, wherein the flexible linker is at least 15 Å in length.
- 26. The composition of claim 1, wherein the flexible linker is at least 28 Å in length.
- 27. The composition of claim 1, wherein the amount of transcription initiated on a linear double-stranded DNA template differs by at least ten-fold compared to a second transcription amount initiated in the absence of the composition.
- 28. The composition of claim 1, wherein the amount of transcription initiated on a linear double-stranded DNA template differs by thirty to fifty-fold compared to the second transcription amount in the absence of the composition.
- 29. The composition of claim 1, wherein the gene comprises a eukaryotic gene.
- 30. A composition for activating transcription having the structure A-B-C, wherein A is a triplex-forming nucleic acid, B is a flexible linker, and C is an effector moiety, wherein B is covalently linked to A and C.
- 31. The composition of claim 30, wherein B is a polyglycol chain, and the covalent linkage of B to C includes a bifunctional crosslinking agent.
- 32. The composition of claim 30, wherein B comprises a polyglycol which is at least about 28 Å in length, and C comprises an amino acid sequence from herpes simplex virus protein VP16.
- 33. A method for assaying a test composition for activity as a transcriptional modulator, comprising:
providing a cell comprising a target gene under the control of at least one transcriptional regulatory element; providing a test composition comprising a DNA binding domain, a test moiety of interest and a flexible linker, one end of the linker being covalently bound to the DNA binding domain, and the other end of the linker being covalently bound to the test compound, the DNA binding domain having affinity for a DNA binding site on a promoter of the target gene sufficient to bind the site; contacting the cell with a test composition; and detecting any changes in the level of transcriptional activity of the target gene in the presence of the test composition compared to the level in the absence of the test composition, which is a measure of the activity of the test composition as a transcriptional modulator.
- 34. A method for assaying a test composition for activity as a transcriptional modulator, comprising:
providing a test composition comprising a DNA binding domain, a test moiety of interest and a flexible linker, one end of the linker being covalently bound to the DNA binding domain, and the other end of the linker being covalently bound to the test compound, the DNA binding domain having affinity for a DNA binding site on a DNA template sufficient to bind the site; contacting the test composition with a transcription mixture comprising a DNA template, a eukaryotic RNA polymerase molecule capable of forming a complex with the test composition and the DNA template, a buffer and substrates under conditions suitable for RNA synthesis, and a detection system for quantitation of RNA product, such that RNA is synthesized; and, detecting any changes in the level of transcriptional activity of the target gene in the presence of the test composition compared to the level in the absence of the test composition, which is a measure of the activity of the test composition as a transcriptional modulator.
- 35. The method of claim 33 or 34, wherein the test moiety is a chemical moiety having a molecular weight of less than about 3 kD.
- 36. The method of claim 33 or 34, wherein the test moiety is a chemical moiety having a molecular weight of less than about 1.5 kD.
- 37. The method of claim 33, wherein the test moiety is a cell membrane-permeant.
- 38. The method of claim 33 or 34, wherein the test moiety is suspected of having transcriptional activation or repressor activity.
- 39. The method of claim 33 or 34, wherein the changes in transcriptional activity are detected as variations in observed levels of mRNA, or protein product encoded by the target gene.
- 40. The method of claim 33 or 34, wherein the target gene is selected from the group consisting of a gene encoding a protein conferring resistance to a drug, a gene encoding an enzyme, a gene which rescues an auxotrophic phenotype, and a gene encoding a cell surface antigen.
- 41. The method of claim 40, wherein the target gene encodes a protein which provides for calorimetric, luminescent or fluorescent detection.
- 42. The method of claim 33 or 34, wherein the step of providing the test composition is performed using high throughput screening technologies comprising robotized sample distribution into multiwell dishes.
- 43. The method of claim 33 or 34, wherein the step of detecting changes in transcriptional activity is performed using the high throughput methods of detection wherein automated plate readers have programmable computerized programs for data analysis and display.
- 44. The method of claim 33 or 34, wherein a plurality of test compositions is provided.
- 45. A method of altering transcription of a target gene, comprising:
introducing into a cell a composition comprising a DNA binding domain having an affinity for a promoter of the target gene, a flexible linker, and a transcriptional effector, one end of the linker being covalently bound to the DNA binding domain, and the other end of the linker being covalently bound to the transcriptional effector.
- 46. The method of claim 45, wherein the effector is an activator or a repressor.
- 47. The method of claim 45, wherein the DNA binding domain is a non-peptidic binding domain.
- 48. A kit for assaying a test composition for activity as a transcriptional modulator, comprising:
a flexible linker covalently bound to a DNA binding domain, the DNA binding domain having affinity for a DNA binding site on a DNA template sufficient to bind the site and activate transcription at a promoter; and a transcription mixture comprising a DNA template and a eukaryotic RNA polymerase molecule that forms a complex with the DNA template.
- 49. The kit of claim 48, wherein the modulator is an activator or a repressor.
- 50. A composition for modulating transcription of a gene, comprising:
a DNA binding domain, a transcriptional effector, one end of the linker being covalently bound to the DNA binding domain, and the other end of the linker being covalently bound to the transcriptional effector, and a non-peptidic flexible linker.
- 51. The method of claim 45, wherein the transcriptional effector is a polypeptide sequence.
- 52. The method of claim 51, wherein the amino terminus of the polypeptide sequence is covalently bound to the linker.
- 53. The method of claim 45, wherein the transcriptional activator is a polypeptide sequence, and the polypeptide sequence comprises at least one copy of an activator sequence of amino acids from herpes virus protein VP 16 selected from the group consisting of comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:8 and SEQ ID NO:12.
- 54. The method of claim 45, wherein the transcriptional effector is a non-peptidic organic moiety.
- 55. The method of claim 45, wherein the transcriptional effector moiety has a molecular weight of less than about 3,000 daltons.
- 56. The method of claim 45, wherein the transcriptional effector moiety has a molecular weight of less than about 1,500 daltons.
- 57. The method of claim 45, wherein the flexible linker comprises a polyglycol.
- 58. The method of claim 57, wherein the flexible linker polyglycol contains at least six glycol units.
- 59. The method of claim 45, wherein the flexible linker comprises one or more units selected from the group consisting of nucleotides, peptides, lower alkyls and oxygen-containing alkyl groups.
- 60. The method of claim 45, wherein the flexible linker is at least 15 Å in length.
- 61. The method of claim 45, wherein the flexible linker is at least 28 Å in length.
- 62. The method of claim 45, wherein the amount of transcription initiated on a linear double-stranded DNA template differs by at least ten-fold compared to a second transcription amount initiated in the absence of the composition.
- 63. The method of claim 45, wherein the amount of transcription initiated on a linear double-stranded DNA template differs by thirty to fifty-fold compared to the second transcription amount in the absence of the composition.
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The application claims priority under 35 U.S.C. §119(e) from provisionally filed application U.S. S. No. 60/240,479, filed on Oct. 13, 2000, and entitled “Artificial Transcriptional Factors and Methods of Use.”
[0002] This application is a continuing application of co-pending application Ser. No. 09/977,852, filed on Oct. 15, 2001, entitled “Artificial Transcriptional Factors and Methods of Use”.
GOVERNMENT FUNDING
[0003] This invention was made in part with government support under grant 1R41 GM57712-01 awarded to the inventor by the National Institute of General Medical Sciences. The government has certain rights in the invention.
Provisional Applications (1)
|
Number |
Date |
Country |
|
60240479 |
Oct 2000 |
US |
Continuations (1)
|
Number |
Date |
Country |
Parent |
09977852 |
Oct 2001 |
US |
Child |
10097800 |
Mar 2002 |
US |