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Details for Patent: 5,475,096

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Details for Patent: 5,475,096

Title: Nucleic acid ligands
Abstract:A new class of nucleic acid compounds, referred to as nucleic acid ligands, have been shown to exist that have a specific binding affinity for three dimensional molecular targets. In a preferred embodiment the nucleic acid ligands are identified by the method of the invention referred to as the Systematic Evolution of Ligands by EXponential enrichment (SELEX), wherein a candidate mixture of nucleic acids are iteratively enriched in high affinity nucleic acids and amplified for further partitioning.
Inventor(s): Gold; Larry (Boulder, CO), Tuerk; Craig (Boulder, CO)
Assignee: University Research Corporation (Boulder, CO)
Filing Date:Jun 10, 1991
Application Number:07/714,131
Claims:1. A non-naturally occurring nucleic acid ligand having a specific binding affinity for a target molecule, such target molecule being a three dimensional chemical structure other than a polynucleotide that binds to said nucleic acid ligand through a mechanism which predominantly depends on Watson/Crick base pairing or triple helix binding, wherein said nucleic acid ligand is not a nucleic acid having the known physiological function of being bound by the target molecule, wherein said target molecule is a protein selected from the group consisting of nucleic acid polymerase, bacteriophage coat protein, serine protease, mammalian receptor, mammalian hormone, mammalian growth factor, ribosomal protein, and viral rev protein.

2. The nucleic acid ligand of claim 1 wherein said nucleic acid polymerase is a DNA polymerase.

3. The nucleic acid ligand of claim 2 wherein said DNA polymerase is gp 43.

4. The nucleic acid ligand of claim 3 consisting of the RNA sequence:

or the corresponding DNA sequence thereof or the corresponding fully complementary sequences thereof.

5. The nucleic acid ligand of claim 1 wherein said nucleic acid polymerase is a reverse transcriptase.

6. The nucleic acid ligand of claim 5 wherein said reverse transcriptase is HIV-1 reverse transcriptase.

7. The nucleic acid ligand of claim 6 wherein said ligand is configured as a pseudoknot, wherein said pseudoknot is comprised of a stem 1(a), a stem 1(b), a stem 2(a), a stem 2(b), a loop 1, a loop 3 and optionally a loop 2, as shown in FIG. 31.

8. The nucleic acid ligand of claim 7 consisting of the RNA sequence 5'-UUCCG-3'.

9. The nucleic acid ligand of claim 7 consisting of the RNA sequence: ##STR81## or the corresponding DNA sequences thereof or the corresponding fully complementary sequence thereof.

10. The nucleic acid ligand of claim 7 consisting of the RNA sequence:

11. The nucleic acid ligand of claim 7 wherein loop 1 is two nucleotides long.

12. The nucleic acid ligand of claim 7 wherein stem 2 is 5 or 6 base pairs.

13. The nucleic acid ligand of claim 7 wherein if loop 2 exists, the nucleotides are adenines.

14. The nucleic acid ligand of claim 7 wherein loop 3 comprises at least 3 nucleotides, said nucleotides enriched in adenine.

15. The nucleic acid ligand of claim 7 wherein loop 1, stem 2(a) and loop 2 consist of eight nucleotides.

16. The nucleic acid ligand of claim 1 wherein said protein is a bacteriophage coat protein.

17. The nucleic acid ligand of claim 16 wherein said bacteriophage coat protein is bacteriophage R17 coat protein.

18. The nucleic acid ligand of claim 17 consisting of the RNA sequence: ##STR82## or the corresponding DNA sequence thereof or the corresponding fully complementary sequences thereof.

19. The nucleic acid ligand of claim 1 wherein said protein is a serine protease.

20. The nucleic acid ligand of claim 19 wherein said serine protease is human tissue plasminogen activator.

21. The nucleic acid ligand of claim 11 wherein said protein is a mammalian receptor.

22. The nucleic acid ligand of claim 21 wherein said mammalian receptor is human growth hormone receptor.

23. The nucleic acid ligand of claim 1 wherein said protein is a mammalian hormone or growth factor.

24. The nucleic acid ligand of claim 23 wherein said mammalian hormone is insulin.

25. The nucleic acid ligand of claim 1 wherein said protein is not known to bind nucleic acids.

26. The nucleic acid ligand of claim 23 wherein said mammalian growth factor is Nerve Growth Factor.

27. The nucleic ligand of claim 26 consisting of a RNA sequence selected from the group consisting of: ##STR83## or the corresponding DNA sequences thereof or the corresponding fully complementary sequences thereof.

28. The nucleic acid ligand of claim 2 wherein said DNA polymerase is HSV-1 DNA polymerase.

29. The nucleic acid ligand of claim 28 consisting of the RNA sequence 5'-UAAGGAGGCCAC-3' (SEQ ID NO:340) or the corresponding DNA sequence thereof or the corresponding fully complementary sequences thereof.

30. The nucleic acid ligand of claim 1 wherein said protein is a ribosomal protein.

31. The nucleic acid ligand of claim 30 wherein said ribosomal protein is E. coli ribosomal protein S1.

32. The nucleic acid ligand of claim 24 consisting of the RNA sequence: ##STR84## or the corresponding DNA sequence thereof or the corresponding fully complementary sequences thereof.

33. The nucleic acid ligand of claim 1 wherein said protein is a viral rev protein.

34. The nucleic acid ligand of claim 33 wherein said viral rev protein is HIV-1 rev protein.

35. The nucleic acid ligand of claim 34 configured as an asymmetric bulge.

36. The nucleic acid ligand of claim 35 consisting of RNA sequences selected from the group consisting of: ##STR85## or the corresponding DNA sequences thereof or the fully complementary sequences thereof.

37. The nucleic acid ligand of claim 1 additionally including a label.

38. The nucleic acid ligand of claim 1 additionally including an antibody or antibody fragment.

39. The nucleic acid ligand of claim 1 which is a nucleic acid selected from the group consisting of single-stranded RNA, double-stranded RNA, single-stranded DNA and double-stranded DNA.

40. The nucleic acid ligand of claim 39 wherein said nucleic acid includes modifications at the cytosine exocyclic amine.

41. The nucleic acid ligand of claim 39 wherein said nucleic acid includes 5'-bromo-uracil substitutions.

42. The nucleic acid ligand of claim 39 wherein said nucleic acid includes backbone modifications.

43. The nucleic acid ligand of claim 39 wherein said nucleic acid includes modifications via methylation.
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