RNA ribozyme polymerases, dephosphorylases, restriction endoribonucleases and methods
First Claim
1. An enzymatic RNA molecule not naturally occurring in nature having an endonuclease activity independent of any protein, said endonuclease activity being 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.
2 Assignments
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Accused Products
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 ribosomal 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.
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Citations
63 Claims
- 1. An enzymatic RNA molecule not naturally occurring in nature having an endonuclease activity independent of any protein, said endonuclease activity being 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.
- 2. A purified enzymatic RNA molecule separated from other RNA molecules and oligonucleotides, said enzymatic RNA molecule having an endonuclease activity independent of any protein, wherein said activity causes cleavage of a separate RNA molecule at a specific nucleotide sequence defining a cleavage site comprising single-stranded RNA in said separate RNA molecule by a transesterification reaction.
- 3. An enzymatic RNA molecule not naturally occurring in nature having an endonuclease activity independent of any protein, said endonuclease activity being specific for a nucleotide sequence defining a cleavage site consisting essentially of a single-stranded region of a separate RNA molecule.
- 4. A purified enzymatic RNA molecule separated from other RNA molecules and oligonucleotides, said enzymatic RNA molecule having an endonuclease activity independent of any protein, wherein said activity causes cleavage of a separate RNA molecule at a specific nucleotide sequence defining a cleavage site consisting essentially of a single-stranded region in said separate RNA molecule.
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22. A method for specifically cleaving a separate RNA molecule at a cleavage site comprising single-stranded RNA, comprising the steps of:
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providing an enzymatic RNA molecule not naturally occurring in nature having an endonuclease activity independent of any protein, said endonuclease activity being specific for a nucleotide sequence defining a cleavage site comprising single-stranded RNA in said separate RNA molecule, and causing cleavage at said cleavage site by a transesterification reaction, and contacting said enzymatic RNA molecule with said separate RNA molecule to cause specific cleavage of said separate RNA molecule at said cleavage site by said endonuclease activity of said enzymatic RNA molecule. - View Dependent Claims (27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 60)
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23. A method for specifically cleaving a separate RNA molecule at a cleavage site comprising single-stranded RNA, comprising the steps of:
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providing a purified enzymatic RNA molecule separated from other RNA molecules and oligonucleotides, said enzymatic RNA molecule having an endonuclease activity independent of any protein, wherein said activity causes cleavage of said separate RNA molecule at a specific nucleotide sequence, defining said cleavage site, by a transesterification reaction, and contacting said enzymatic RNA molecule with said separate RNA molecule to cause specific cleavage of said separate RNA molecule at said cleavage site by said endonuclease activity of said enzymatic RNA molecule.
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24. A method for specifically cleaving a separate RNA molecule at a cleavage site comprising single-stranded RNA, comprising the steps of:
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providing an enzymatic RNA molecule not naturally occurring in nature and having an endonuclease activity independent of any protein, said endonuclease activity being specific for a said cleavage site consisting essentially of a single-stranded region of said separate RNA molecule, and contacting said enzymatic RNA molecule with said separate RNA molecule to cause specific cleavage of said separate RNA molecule at said cleavage site by said endonuclease activity of said enzymatic RNA molecule. - View Dependent Claims (26)
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25. A method for specifically cleaving a separate RNA molecule comprising a cleavage site comprising single-stranded RNA, at the steps of:
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providing a purified enzymatic RNA molecule separated from other RNA molecules and oligonucleotides, said enzymatic RNA molecule having an endonuclease activity independent of any protein, wherein said activity causes cleavage of said separate RNA molecule at a said cleavage site consisting essentially of a single-stranded region in said separate RNA molecule, and contacting said enzymatic RNA molecule with said separate RNA molecule to cause specific cleavage of said separate RNA molecule at said cleavage site by said endonuclease activity of said enzymatic RNA molecule.
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- 43. A purified enzymatic RNA molecule having an endonuclease activity independent of any protein, wherein said activity causes cleavage of a separate RNA molecule at a specific nucleotide sequence defining a cleavage site consisting essentially of single-stranded RNA in said 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.
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45. A method for specifically cleaving a separate RNA molecule at a cleavage site comprising single-stranded RNA, comprising the steps of:
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providing an enzymatic RNA molecule having an endonuclease activity independent of any protein, wherein said activity causes cleavage of a separate RNA molecule at a specific nucleotide sequence defining a cleavage site consisting essentially of single-stranded RNA in a separate RNA molecule, and contacting said enzymatic RNA molecule and said separate RNA molecule to cause specific cleavage of said separate RNA molecule at said cleavage site by said endonuclease activity of said enzymatic RNA molecule. - View Dependent Claims (46, 47, 61)
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- 48. A purified enzymatic RNA molecule, separated from other RNA molecules and oligonucleotides, having endonuclease activity independent of any protein, said activity being specific for a nucleotide sequence defining a cleavage site comprising single-stranded RNA in a separate RNA molecule, said enzymatic RNA molecule being synthesized from DNA.
- 51. An enzymatic RNA molecule having an endonuclease activity independent of any protein, said activity being specific for a nucleotide sequence defining a cleavage site comprising single-stranded RNA in a separate RNA molecule, said enzymatic RNA molecule being chemically synthesized to correspond to naturally occurring RNA wherein the naturally-occurring cleavage site of said naturally-occurring RNA has been deleted.
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53. A method for producing an enzymatic RNA molecule having an endonuclease activity independent of any protein, said activity being specific for a nucleotide sequence defining a cleavage site comprising single-stranded RNA in a separate first RNA molecule, comprising the steps of:
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identifying a second RNA molecule having intramolecular cleaving activity, determining the cleavage site of said second RNA molecule, said site comprising at least four bases, and synthesizing an enzymatic RNA molecule corresponding to said second RNA molecule lacking said cleavage site. - View Dependent Claims (54, 55, 56)
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- 57. An enzymatic RNA molecule, isolated from its natural environment, having an endonuclease activity independent of any protein, wherein said activity causes cleavage of a separate RNA molecule at a specific nucleotide sequence defining a cleavage site comprising single-stranded RNA in said 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.
Specification