Hyperstabilizing antisense nucleic acid binding agents

Abstract

Attaching certain ligands to antisense probes will hyperstabilize sense-antisense duplexes. Such a hyperstabilized duplex is resistant to melting of the strands from one another, to unwinding of the strands, and to the action of nucleases. Applications include antiretroviral action, antireverse-transcriptase action, antiviral action, antiparasitical action, antibacterial action, antifungal action, anticancer action, anti-oncogene action, and other applications where it is desired to inhibit gene expression at the genomic or messenger RNA level. The preferred ligands are certain minor-groove-binding agents, exemplified by CC-1065 and synthetic CC-1065 analogs.

I claim:  
     
       1. A composition for binding a selected ribonucleic acid sequence which is not a homopolymer,  comprising: 
       (a) an oligonucleotide which is not a homopolymer, and which is complementary to the  a selected ribonucleic acid sequence which is not a homopolymer, and which can base-pair to the selected ribonucleic acid sequence to form a double helix having a minor groove and a major groove; and  
       (b) a non-alkylating binding agent which preferentially binds in the minor groove of a double helix formed by the base-pairing of said oligonucleotide and the selected ribonucleic acid sequence, and which increases the strand separation temperature of the double helix; wherein said binding agent is selected from the group consisting of CC- 1065 ,  tristabilin, an agent comprising a pyrrolomethoxy hydroxyindole  from two to four pyrrolomethoxyhydroxyindole tricyclic ring, U 71 , 184 , an indole, an indole  rings, an agent comprising from two to four indoles, an agent comprising from two to four indoles substituted with at least one hydroxy group, an indole  agent comprising from two to four indoles substituted with at least one methoxy group, an indole  and an agent comprising from two to four indoles substituted with at least one pyrrole, and a lexitropsin .  
     
     
       2. A composition for binding a selected ribonucleic acid sequence which is not a homopolymer, comprising: 
       (a) an oligonucleotide which is not a homopolymer, and which is complementary to the selected ribonucleic acid sequence, and which can base-pair to the selected ribonucleic acid sequence to form a double helix having a minor groove and a major groove; and  
       (b) a binding agent which preferentially binds in the minor groove of a double helix formed by the base-pairing of said oligonucleotide and the selected ribonucleic acid sequence, and which increases the strand separation temperature of the double helix, wherein said binding agent is chemically tethered to said oligonucleotide.  
     
     
       3. A composition as recited in  claim 1 , wherein said binding agent comprises CC- 1065 . 
     
     
       4. A composition as recited in claim  3 ,   1 , wherein said binding agent is chemically tethered to said oligonucleotide. 
     
     
       5. A composition as recited in  claim 4 , wherein said binding agent and said oligonucleotide are each covalently bound to succinimidyl 6-(6-(((iodoacetyl) amino) hexanoyl) amino) hexanoate. 
     
     
       6. A composition as recited in  claim 1 , wherein said binding agent comprises tristabilin. 
     
     
       7. A composition as recited in  claim 6 , wherein said binding agent is chemically tethered to said oligonucleotide. 
     
     
       8. A composition as recited to  claim 1 , wherein said binding agent comprises a pyrrolomethoxyhydroxyindole tricyclic ring. 
     
     
       9. A composition as recited in  claim 8 , wherein said binding agent is chemically tethered to said oligonucleotide. 
     
     
       10. A composition as recited in claim  8 ,   1 , wherein said binding agent comprises three pyrrolomethoxyhydroxyindole tricyclic rings. 
     
     
       11. A composition as recited in  claim 10 , wherein said binding agent is chemically tethered to said oligonucleotide. 
     
     
       12. A composition as recited in claim  8 ,   1 , wherein said binding agent comprises two pyrrolomethoxyhydroxyindole tricyclic rings. 
     
     
       13. A composition as recited in  claim 12 , wherein said binding agent is chemically tethered to said oligonucleotide. 
     
     
       14. A composition as recited in claim  8 ,   1 , wherein said binding agent comprises four pyrrolomethoxyhydroxyindole tricyclic rings. 
     
     
       15. A composition as recited in  claim 14 , wherein said binding agent is chemically tethered to said oligonucleotide. 
     
     
       16. A composition as recited in  claim 1 , wherein said binding agent comprises U- 71 , 184 . 
     
     
       17. A composition as recited in  claim 16 , wherein said binding agent is chemically tethered to said oligonucleotide. 
     
     
       18. A composition as recited in  claim 1 , wherein said binding agent comprises an indole, or an indole  from two to four indoles, or from two to four indoles substituted with at least one functional group selected from the group consisting of hydroxy, methoxy, and pyrrole. 
     
     
       19. A composition as recited in  claim 18 , wherein said binding agent is chemically tethered to said oligonucleotide. 
     
     
       20. A composition as recited in  claim 1 , wherein said binding agent comprises a lexitropsin. 
     
     
       21. A composition as recited in claim  20 ,   1 , wherein said binding agent comprises a lexitropsin, and wherein said binding agent is chemically tethered to said oligonucleotide. 
     
     
       22. A composition for binding a selected ribonucleic acid sequence which is not a homopolymer, comprising:  as recited in  claim 1 ,  
       (a) an oligonucleotide which is not a homopolymer, and which is complementary to the selected ribonucleic acid sequence, and which can base-pair to the selected ribonucleic acid sequence to form a double helix having a minor groove and a major groove,  wherein said oligonucleotide contains internucleotide bonds which comprise phosphorothioate or methyl phosphonate; and  
       (b) a binding agent which preferentially binds in the minor groove of a double helix formed by the base-pairing of said oligonucleotide and the selected ribonucleic acid sequence, and which increases the strand separation temperature of the double helix .  
     
     
       23. A composition as recited in  claim 22 , wherein said binding agent is chemically tethered to said oligonucleotide. 
     
     
       24. An in vitro method for binding a selected ribonucleic acid sequence which is not a homopolymer, comprising the steps of: 
       (a) contacting the selected ribonucleic acid sequence in vitro with an oligonucleotide which is not a homopolymer, and which is complementary to the selected ribonucleic acid sequence, and which can base-pair to the selected ribonucleic acid sequence to form a double helix having a minor groove and a major groove, until such a double helix is formed; and  
       (b) contacting the double helix in vitro with a non-alkylating binding agent which preferentially binds in the minor groove of the double helix, and which increases the strand separation temperature of the double helix, until said binding agent has bound in the minor groove; wherein the binding agent is selected from the group consisting of tristabilin, an agent comprising from two to four pyrrolomethoxyhydroxyindole tricyclic rings, an agent comprising from two to four indoles, an agent comprising from two to four indoles substituted with at least one hydroxy group, an agent comprising from two to four indoles substituted with at least one methoxy group, and an agent comprising from two to four indoles substituted with at least one pyrrole.  
     
     
       25. A method as recited in  claim 24 , wherein said binding agent is chemically tethered to said oligonucleotide. 
     
     
       26. A method as recited in  claim 24 , wherein said binding agent comprises CC- 1065 . 
     
     
       27. A method as recited in  claim 26 , wherein said binding agent is chemically tethered to said oligonucleotide. 
     
     
       28. A method as recited in  claim 27 , wherein said binding agent and said oligonucleotide are each covalently bound to succinimidyl  6 -( 6 -(((iodacetyl) amino) hexanoyl) amino) hexanoate. 
     
     
       29. A method as recited in  claim 24 , wherein said binding agent comprises tristabilin. 
     
     
       30. A method as recited in  claim 29 , wherein said binding agent is chemically tethered to said oligonucleotide. 
     
     
       31. A method as recited in claim  30 ,   24 , wherein said binding agent comprises a  from two to four pyrrolomethoxyhydroxyindole tricyclic ring  rings. 
     
     
       32. A method as recited in  claim 31 , wherein said binding agent is chemically tethered to said oligonucleotide. 
     
     
       33. A method as recited in  claim 31 , wherein said binding agent comprises three pyrrolomethoxyhydroxyindole tricyclic rings. 
     
     
       34. A method as recited in  claim 33 , wherein said binding agent is chemically tethered to said oligonucleotide. 
     
     
       35. A method as recited in  claim 31 , wherein said binding agent comprises two pyrrolomethoxyhydroxyindole tricyclic rings. 
     
     
       36. A method as recited in  claim 35 , wherein said binding agent is chemically tethered to said oligonucleotide. 
     
     
       37. A method as recited in  claim 31 , wherein said binding agent comprises four pyrrolomethoxyhydroxyindole tricyclic rings. 
     
     
       38. A method as recited in  claim 37 , wherein said binding agent is chemically tethered to said oligonucleotide. 
     
     
       39. A method as recited in  claim 24 , wherein said binding agent comprises U- 71 , 184 . 
     
     
       40. A method as recited in  claim 39 , wherein said binding agent is chemically tethered to said oligonucleotide. 
     
     
       41. A method as recited in  claim 24 , wherein said binding agent comprises an indole, or an indole  from two to four indoles, or from two to four indoles substituted with at least one functional group selected from the group consisting of hydroxy, methoxy and pyrrole. 
     
     
       42. A method as recited in  claim 41 , wherein said binding agent is chemically tethered to said oligonucleotide. 
     
     
       43. A method as recited in  claim 24 , wherein said binding agent comprises a lexitropsin. 
     
     
       44. A method as recited in  claim 43 , wherein said binding agent is chemically tethered to said oligonucleotide. 
     
     
       45. A method as recited in  claim 24 , wherein said oligonucleotide contains internucleotide bonds which comprise phosphorothioate or methyl phosphonate. 
     
     
       46. A method as recited in  claim 45 , wherein said binding agent is chemically tethered to said oligonucleotide. 
     
     
       47. A composition of matter comprising tristabilin. 
     
     
       48. A composition as recited in  claim 4 , wherein said binding agent is chemically tethered to said oligonucleotide at the  3 ′ end of said oligonucleotide. 
     
     
       49. A composition as recited in  claim 4 , wherein said binding agent is chemically tethered to said oligonucleotide at the  5 ′ end of said oligonucleotide. 
     
     
       50. A composition as recited in  claim 4 , wherein said binding agent is chemically tethered to said oligonucleotide at an internal site of said oligonucleotide. 
     
     
       51. A method as recited in  claim 24 , wherein said binding agent is chemically tethered to said oligonucleotide at the  3 ′ end of said oligonucleotide. 
     
     
       52. A method as recited in  claim 24 , wherein said binding agent is chemically tethered to said oligonucleotide at the  5 ′ end of said oligonucleotide. 
     
     
       53. A method as recited in  claim 24 , wherein said binding agent is chemically tethered to said oligonucleotide at an internal site of said oligonucleotide.