RNA ribozyme restriction endoribonucleases and methods
AssigneeUniversity Patents, Inc.
InventorsThomas R. Cech, Felicia L. Murphy, Arthur J. Zaug, Cheryl Grosshans
Life of this patent
Estimating the term…
Abstract
New RNA endoribonuclease ribozymes are found with new conditions to prevent mismatch cleavage and able to cleave RNA after 6 different sets of ribonucleotide 4 base sequences.
Claims22
A method for preventing non-complementary cleavage by an enzymatic RNA molecule of a separate RNA molecule, comprising the steps of:
- providing an enzymatic RNA molecule having an RNA substrate-binding site comprising a first nucleotide sequence and an endonuclease activity independent of any protein in vitro, said endonuclease activity being active at or at a location proximal to a second nucleotide sequence in the separate RNA molecule complementary to said first nucleotide sequence, and
- contacting said enzymatic RNA molecule and the separate RNA molecule in the presence of a denaturing agent to allow said endonuclease activity to cause specific cleavage of said separate RNA molecule at or proximal to said second nucleotide sequence but not at any other nucleotide sequence which is not complementary to said first nucleotide sequence.
A method for cleaving a cleavage site in a separate RNA molecule, comprising the steps of:
- providing an enzymatic RNA molecule comprising an RNA substrate-binding site formed from at least four nucleotides, said enzymatic RNA molecule having an endonuclease activity independent of any protein in vitro, said endonuclease activity being active at or at a site proximal to a nucleotide sequence complementary to said at least four nucleotides at the RNA substrate-binding site of said enzymatic molecule,
- providing the separate RNA molecule, wherein said cleavage site in the separate RNA molecule is complementary to said at least four nucleotides at at least two of said four nucleotides;
- and
- contacting said enzymatic RNA molecule and the separate RNA molecule to allow said endonuclease activity to cleave said separate RNA molecule at or proximal to said cleavage site.
A method for cleaving a cleavage site in a separate RNA molecule, comprising the steps of:
- providing an enzymatic RNA molecule comprising an RNA substrate-binding site formed from at least five nucleotides, said enzymatic RNA molecule having an endonuclease activity independent of any protein in vitro, said endonuclease activity being active at or at a site proximal to a nucleotide sequence complementary to said at least five nucleotides at the RNA substrate-binding site of said enzymatic molecule,
- providing the separate RNA molecule, wherein said cleavage site in the separate RNA molecule is complementary to said at least five nucleotides at at least two of said five nucleotides;
- and
- contacting said enzymatic RNA molecule and the separate RNA molecule to allow said endonuclease activity to cleave said separate RNA molecule at or proximal to said cleavage site.
A method for cleaving a cleavage site in a separate RNA molecule, comprising the steps of:
- providing an enzymatic RNA molecule comprising an RNA substrate-binding site formed from at least six nucleotides, said enzymatic RNA molecule having an endonuclease activity independent of any protein in vitro, said endonuclease activity being active at or at a site proximal to a nucleotide sequence complementary to said at least six nucleotides at the RNA substrate-binding site of said enzymatic molecule,
- providing the separate RNA molecule, wherein said cleavage site in the separate RNA molecule is complementary to said at least six nucleotides at at least two of said six nucleotides;
- and
- contacting said enzymatic RNA molecule and the separate RNA molecule to allow said endonuclease activity to cleave said separate RNA molecule at or proximal to said cleavage site.
A DNA vector comprising DNA encoding L-21 ScaI enzymatic RNA, said DNA being constructed and arranged to cause production of the L-21 ScaI enzymatic RNA molecule when contacted with ScaI restriction endonuclease and an RNA polymerase under transcriptional conditions.
Dependencies are inferred from the claim text.