RNA Ribozyme which cleaves substrate RNA without formation of a convalent bond

• US 5,354,855 A
• Filed: 02/28/1992
• Issued: 10/11/1994
• Est. Priority Date: 12/03/1986
• Status: Expired due to Term

• Current Assignee: Arthur J. Zaug, Michael D. Been, Thomas R. Cech
• Original Assignee: Arthur J. Zaug, Michael D. Been, Thomas R. Cech
• Inventors: Cech, Thomas R., Zaug, Arthur J., Been, Michael D.
• Primary Examiner(s): Schwartz, Richard A.
• Assistant Examiner(s): Choi, Kathleen L.
• Application Number:
  US07/843,737
• Time in Patent Office: 956 Days
• Field of Search: 435/91.1, 435/172.3, 435/193, 435/194, 435/320.1, 435/258, 935/3, 935/14, 935/16, 536/23.1, 536/24.2
• US Class Current: 536/24.1
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 2310/111 spanning the whole gene, or a large part of it
• CPC Class Codes: C12N 2310/1241 Tetrahymena

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.

Claims

1. A purified enzymatic ribonucleic acid molecule having an endoribonuclease activity independent of any protein, said endoribonuclease activity acting on a nucleotide sequence defining a cleavage site in a separate RNA molecule and causing cleavage at said cleavage site by a transesterification reaction;

wherein said enzymatic ribonucleic acid molecule does not form a covalent bond with said separate RNA molecule during said cleavage.