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Last Updated: September 22, 2021

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Claims for Patent: 5,093,246

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Summary for Patent: 5,093,246
Title: RNA ribozyme polymerases, dephosphorylases, restriction endoribo-nucleases and methods
Abstract:RNA enzymes or ribozymes can act as endoribonucleases, catalyzing the cleavage of RNA molecules with a sequence specificity of cleavage greater than that of known ribonucleases and approaching that of the DNA restriction endonucleases, thus serving as RNA sequence specific endoribonucleases. An example is a shortened form of the self-splicing ribonsomal RNA intervening sequence of Tetrahymena (L-19 IVS RNA). Site-specific mutagenesis of the enzyme active site of the L-19 IVS RNA alters the substrate sequence specificity in a predictable manner, allowing a set of sequence-specific endoribonucleases to be synthesized. Varying conditions allow the ribozyme to act as a polymerase (nucleotidyltransferase), a dephosphorylase (acid phosphatase or phosphotransferase) or a sequence-specific endoribonuclease.
Inventor(s): Cech; Thomas R. (Boulder, CO), Zaug; Arthur J. (Louisville, CO), Been; Michael D. (Boulder, CO)
Assignee: University Patents, Inc. (Westport, CT)
Application Number:07/562,672
Patent Claims:1. Method for dephosphorylation of an RNA substrate comprising the steps of:

providing an enzymatic RNA molecule having a dephosphorylation activity independent of any protein, and

contacting said enzymatic RNA molecule with said RNA substrate under conditions suitable for said enzymatic RNA molecule to remove the 3' terminal phosphate of said RNA substrate.

2. A method for locating a nucleotide sequence in an RNA molecule having a first length, comprising the steps of:

providing an enzymatic RNA molecule having an endonuclease activity independent of any protein,

contacting said enzymatic RNA molecule with said RNA molecule wherein said enzymatic RNA molecule will cleave said RNA molecule at any said nucleotide sequence present in said RNA molecule, and

determining the length of resulting RNA molecules resulting from said contacting step as an indication of the location of the nucleotide sequence in said RNA molecule.

3. A method for locating a plurality of nucleotide sequences in an RNA molecule having a first length, comprising the steps of:

providing a plurality of different enzymatic RNA molecules having an endonuclease activity independent of any protein, each said different enzymatic RNA molecule cleaving said RNA molecule at a different said nucleotide sequence of said plurality of nucleotide sequences;

contacting said plurality of different enzymatic RNA molecules with said RNA molecule wherein each said different enzymatic RNA molecule will cleave said RNA molecule at any said different nucleotide sequence present in said RNA molecule, and

determining the length of resulting RNA molecules resulting from said contacting step as an indication of the location of the nucleotide sequence in said RNA molecule.

4. A method for detecting the presence of a nucleotide sequence in an RNA molecule, comprising the steps of:

providing an enzymatic RNA molecule having an endonuclease activity independent of any protein,

contacting said enzymatic RNA molecule with said RNA molecule wherein said enzymatic RNA molecule will cleave said RNA molecule of any said nucleotide sequence present in said RNA molecule to produce two or more fragments of said RNA molecule, and

detecting the presence of said fragments as an indication of the presence of said nucleotide sequence.

5. Method of claim 2, 3 or 4 wherein a radiolabelled guanosine derivative is provided in said contacting step to cause radiolabelling of the 5' end of any cleaved said RNA molecule.

6. Method for producing an enzymatic RNA molecule having an endonuclease activity independent of any protein, comprising the steps of:

(a) transcribing recombinant DNA encoding a precursor RNA molecule to said enzymatic RNA molecule with an RNA polymerase in vitro to produce said precursor RNA;

(b) incubating said precursor RNA under conditions suitable for promoting self-splicing and cyclization of said precursor RNA to produce cyclized RNA;

(c) contacting said cyclized RNA with MgCl.sub.2 at pH 9.0 to promote conversion of said cyclized RNA to said enzymatic RNA molecule; and

(d) purifying said enzymatic RNA molecule by polyacrylamide gel electrophoresis and chromatography.

7. Method of claim 6 wherein said recombinant DNA is cloned in E. coli.

8. Method of claim 6 wherein said recombinant DNA comprises an intron of a Tetrahymena gene encoding ribosomal RNA.

9. Method of claim 6 wherein the expression of said DNA is regulated by the SP6 or T7 RNA polymerase promoter.

10. A method for producing an enzymatic RNA molecule having an endonuclease activity of any protein, comprising the steps of:

providing DNA encoding RNA consisting essentially of said enzymatic RNA molecule under the transcriptional control of a promoter, and

transcribing said DNA from said promoter to produce said enzymatic RNA molecule.

11. A vector for production of an enzymatic RNA molecule having an endonuclease activity independent of any protein, comprising:

DNA encoding said enzymatic RNA molecule, and

a promoter positioned relative to said DNA to cause transcription of said DNA to form an RNA molecule consisting essentially of said enzymatic RNA molecule.

12. The vector of claim 11 wherein said endonuclease activity is specific for a nucleotide sequence defining a cleavage site comprising single stranded RNA in a separate RNA molecule and causing cleavage at said cleavage site by a transesterification reaction.

13. The vector of claim 11 wherein said endonuclease activity is specific for a nucleotide sequence defining a cleavage site consisting essentially of a single stranded region of a separate RNA molecule.

14. The vector of claim 11 or 13 wherein the nucleotide sequence defining said cleavage site comprises four or more bases.

15. The vector of claim 13 wherein said endonuclease activity causes cleavage at said cleavage site by a transesterification reaction.

16. The vector of claim 11 wherein said enzymatic RNA molecule comprises an active site region responsible for said cleavage substantially identical to the active site region of the ribosomal RNA intervening sequence naturally occurring in Tetrahymena.

17. The vector of claim 16 wherein said nucleotide sequence defining said cleavage site is chosen from GUCU, CUGU, and CGCU.

18. The vector of claim 11 wherein said enzymatic RNA molecule has a pH optimum for said endonuclease activity between 7.5 and 8.

19. The vector of claim 11 wherein said endonuclease activity requires the presence of magnesium ions, a denaturant, or a guanine derivative.

20. The vector of claim 11 wherein said endonuclease activity is enhanced by the presence of a divalent cation.

21. The vector of claim 20 wherein said divalent cation is magnesium.

22. The vector of claim 11 wherein said enzymatic RNA molecule further comprises a second endonuclease activity, independent of any protein, for nucleic acid comprising deoxyribonucleotides wherein said endonuclease activity and said second endonuclease activity may be the same or different.

23. The vector of claim 11 wherein said enzymatic RNA molecule further has a nucleotidyl transferase activity.

24. The vector of claim 23 wherein said nucleotidyl transferase activity causes polymerization of oligocytidylic acid to polycytidylic acid.

25. The vector of claim 11 wherein said enzymatic RNA molecule further has a dephosphorylase activity.

26. The vector of claim 25 wherein said dephosphorylase activity causes removal of a 3' terminal phosphate of a nucleic acid comprising a 3' ribonucleotide.

27. The vector of claim 26 wherein said dephosphorylase activity is specific to a specific nucleotide sequence.

28. The vector of claim 11 wherein said enzymatic RNA molecule comprises the ribonucleotide base sequence of L-19 RNA, said enzymatic RNA molecule having a ribonucleotide base sequence at the active site responsible for said cleavage different from that of L-19 from Tetrahymena.

29. The vector of claim 11 wherein said enzymatic RNA molecule comprises the ribonucleotide base sequence of L-19 RNA.

30. The vector of claim 29 wherein said L-19 comprises bases 19-331 of Tetrahymena L-IVS.

31. The vector of claim 11 wherein said activity causes cleavage of a separate RNA molecule at a specific nucleotide sequence defining a cleavage site comprising single stranded RNA in a separate RNA molecule, said enzymatic RNA molecule not causing specific cleavage of said separate RNA molecule immediately adjacent in the direction 3' to the nucleotide sequence CUCU.

32. The vector of claim 11, said vector being a plasmid.

33. The vector of claim 11 wherein said promoter is selected from the group consisting of the bacteriophage SP6 and bacteriophage T7 promoters.

34. The vector of claim 11, wherein said enzymatic RNA molecule comprises bases 21-331 of Tetrahymena L-IVS.

35. A plasmid selected from the group consisting of pSPTT1A3, pT7-TT1A3, pBGST7, pBG/-2G:23C, pBG/23C, pBG/-3G:24C, pBG/24C, pBG/-4G:25C, pBG/25C, and pBG/23A.sub.4.

Details for Patent 5,093,246

Applicant Tradename Biologic Ingredient Dosage Form BLA Number Approval Date Patent No. Assignee Estimated Patent Expiration Status Orphan Source
Schering INTRON A interferon alfa-2b VIAL 103132 001 1986-06-04 ⤷  Free Forever Trial University Patents, Inc. (Westport, CT) 2009-03-03 RX search
Schering INTRON A interferon alfa-2b VIAL 103132 002 1986-06-04 ⤷  Free Forever Trial University Patents, Inc. (Westport, CT) 2009-03-03 RX search
Schering INTRON A interferon alfa-2b VIAL 103132 003 1986-06-04 ⤷  Free Forever Trial University Patents, Inc. (Westport, CT) 2009-03-03 RX search
>Applicant >Tradename >Biologic Ingredient >Dosage Form >BLA >Number >Approval Date >Patent No. >Assignee >Estimated Patent Expiration >Status >Orphan >Source

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