University Patents, Inc.

Competitive Technologies, Inc.

Duke University

Other

Michael D Been

Thomas R Cech

Arthur J Zaug

Sarah P Rosenstein

Anne T Perrotta

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.

RNA ribozyme polymerases, dephosphorylases, restriction endoribonucleases and methods

RNA ribozyme polymerases, dephosphorylases, restriction

Nucleic acid molecule having an RNA substrate cleaving enzymatic activity which cleaves a separate RNA substrate at a cleavage site. The nucleic acid molecule includes an RNA substrate binding portion which base pairs with the RNA substrate only 3' of the cleavage site, and an enzymatic portion (which may include a part or all of the RNA substrate binding portion) having the enzymatic activity. The nucleic acid molecule is able to base pair with the RNA substrate only 3' of the cleavage site, and cause cleavage of the RNA substrate at that cleavage site. The nucleic acid molecule can be either linear or circular. A general method for forming circular RNA in vivo and in vitro is provided.

Enzymatic RNA molecules

RNA Ribozyme which cleaves substrate RNA without formation of a

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.

A catalytic RNA (ribozyme) derived from an intervening sequence (IVS) RNA of Tetrahymena thermophila will catalyze an RNA polymerization reaction in which pentacytidylic acid (C.sub.5) is extended by the successive addition of mononucleotides derived from a guanylyl-(3',5')-nucleotide (GpN). Cytidines or uridines are added to C.sub.5 to generate chain lengths of 10 to 11 nucleotides; longer products are also generated but at reduced efficiency. The reaction is analogous to that catalyzed by a replicase with C.sub.5 acting as the primer, GpNs as the nucleoside triphosphates, and a sequence in the ribozyme providing a template.

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Durham, NC, United States of America

Michael D Been