Extending the cleavage rules for the hammerhead ribozyme: mutating adenosine15.1 to inosine15.1 changes the cleavage site specificity from N16.2U16.1H17 to N16.2C16.1H17.

In this paper, we show that an adenosine to inosine mutation at position 15.1 changes the substrate specificity of the hammerhead ribozyme from N16.2U16.1H17to N16.2C16.1H17(H represents A, C or U). This result extends the hammerhead cleavage triplet definition from N16.2U16.1H17to the more general N16.2Y16.1H17. Comparison of cleavage rates using I15.1ribozymes for NCH triplets and standard A15.1 ribozymes for NUH triplets under single turnover conditions shows similar or slightly enhanced levels of reactivity for the I15. 1-containing structures. The effect of I15.1 substitution was also tested in nuclease-resistant 2'- O -alkyl substituted derivatives (oligozymes), showing a similar level of activity for the NUH and NCH cleaving structures. The availability of NCH triplets that can be targeted without loss of efficiency increases the flexibility of ribozyme targeting strategies. This was demonstrated by an efficient cleavage of an HCV transcript at a previously inaccessible GCA site in codon 2.

[1]  T. Campbell,et al.  The effect of structure in a long target RNA on ribozyme cleavage efficiency. , 1997, Nucleic acids research.

[2]  J. F. Atkins,et al.  A rapid in vitro method for obtaining RNA accessibility patterns for complementary DNA probes: correlation with an intracellular pattern and known RNA structures. , 1997, Nucleic acids research.

[3]  R. Ornstein,et al.  Structure of an anti-HIV-1 hammerhead ribozyme complex with a 17-mer DNA substrate analog of HIV-1 gag RNA and a mechanism for the cleavage reaction: 750 MHz NMR and computer experiments. , 1997, Journal of biomolecular structure & dynamics.

[4]  S. Asano,et al.  Comparison of the specificities and catalytic activities of hammerhead ribozymes and DNA enzymes with respect to the cleavage of BCR-ABL chimeric L6 (b2a2) mRNA. , 1997, Nucleic acids research.

[5]  R R Breaker,et al.  Examination of the catalytic fitness of the hammerhead ribozyme by in vitro selection. , 1997, RNA.

[6]  O. Uhlenbeck,et al.  Hammerhead ribozymes with a faster cleavage rate. , 1997, Biochemistry.

[7]  B. Bass,et al.  Detection of inosine in messenger RNA by inosine-specific cleavage. , 1997, Biochemistry.

[8]  E. Southern,et al.  Selecting effective antisense reagents on combinatorial oligonucleotide arrays , 1997, Nature Biotechnology.

[9]  N. Vaish,et al.  Isolation of hammerhead ribozymes with altered core sequences by in vitro selection. , 1997, Biochemistry.

[10]  G. F. Joyce,et al.  A general purpose RNA-cleaving DNA enzyme. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[11]  W. Lima,et al.  Control of complexity constraints on combinatorial screening for preferred oligonucleotide hybridization sites on structured RNA. , 1997, Biochemistry.

[12]  H. Soreq,et al.  Probing accessible sites for ribozymes on human acetylcholinesterase RNA. , 1997, RNA.

[13]  F. Eckstein,et al.  The structure, function and application of the hammerhead ribozyme. , 1997, European journal of biochemistry.

[14]  O. Uhlenbeck,et al.  A kinetic and thermodynamic analysis of cleavage site mutations in the hammerhead ribozyme. , 1997, Biochemistry.

[15]  R. Hanecak,et al.  Combinatorial Screening and Rational Optimization for Hybridization to Folded Hepatitis C Virus RNA of Oligonucleotides with Biological Antisense Activity* , 1997, The Journal of Biological Chemistry.

[16]  B. Stoddard,et al.  Capturing the Structure of a Catalytic RNA Intermediate: The Hammerhead Ribozyme , 1996, Science.

[17]  T. Jarvis,et al.  Optimizing the Cell Efficacy of Synthetic Ribozymes , 1996, The Journal of Biological Chemistry.

[18]  A. Karpeisky,et al.  Chemically modified hammerhead ribozymes with improved catalytic rates. , 1996, Biochemistry.

[19]  D. Mckay,et al.  Structure and function of the hammerhead ribozyme: an unfinished story. , 1996, RNA.

[20]  A. Beaudry,et al.  Inhibition of vascular smooth muscle cell proliferation by ribozymes that cleave c-myb mRNA. , 1996, RNA.

[21]  E. Jankowsky,et al.  Oligonucleotide facilitators may inhibit or activate a hammerhead ribozyme. , 1996, Nucleic acids research.

[22]  H. Prydz,et al.  A synthetic, chemically modified ribozyme eliminates amelogenin, the major translation product in developing mouse enamel in vivo. , 1995, The EMBO journal.

[23]  J. Arnold,et al.  The roles of the conserved pyrimidine bases in hammerhead ribozyme catalysis: evidence for a magnesium ion-binding site. , 1995, The Biochemical journal.

[24]  K. Taira,et al.  Ribozymes: from mechanistic studies to applications in vivo. , 1995, Journal of biochemistry.

[25]  A. Klug,et al.  The crystal structure of an AII-RNAhammerhead ribozyme: A proposed mechanism for RNA catalytic cleavage , 1995, Cell.

[26]  R. Christoffersen,et al.  Ribozymes as human therapeutic agents. , 1995, Journal of medicinal chemistry.

[27]  M. Tabler,et al.  Comparative analysis of cleavage rates after systematic permutation of the NUX consensus target motif for hammerhead ribozymes. , 1995, Nucleic acids research.

[28]  K. Taira,et al.  Generality of the NUX rule: kinetic analysis of the results of systematic mutations in the trinucleotide at the cleavage site of hammerhead ribozymes. , 1995, Biochemistry.

[29]  K. Flaherty,et al.  Three-dimensional structure of a hammerhead ribozyme , 1994, Nature.

[30]  T. Tuschl,et al.  Isoguanosine substitution of conserved adenosines in the hammerhead ribozyme. , 1994, Biochemistry.

[31]  L. McLaughlin,et al.  Importance of specific guanosine N7-nitrogens and purine amino groups for efficient cleavage by a hammerhead ribozyme. , 1993, Biochemistry.

[32]  O. Uhlenbeck,et al.  Kinetics of intermolecular cleavage by hammerhead ribozymes. , 1992, Biochemistry.

[33]  A. Lamond,et al.  Nuclease resistant ribozymes with high catalytic activity. , 1992, The EMBO journal.

[34]  M. Gait,et al.  The role of the exocyclic amino groups of conserved purines in hammerhead ribozyme cleavage. , 1992, Biochemical and biophysical research communications.

[35]  S. Benner,et al.  Synthesis of RNA containing inosine: analysis of the sequence requirements for the 5' splice site of the Tetrahymena group I intron. , 1991, Nucleic acids research.

[36]  G D Stormo,et al.  Sequence requirements of the hammerhead RNA self-cleavage reaction. , 1990, Biochemistry.

[37]  R. Symons,et al.  Self-cleavage of RNA in the replication of small pathogens of plants and animals. , 1989, Trends in biochemical sciences.

[38]  W. Gerlach,et al.  Simple RNA enzymes with new and highly specific endoribonuclease activities , 1988, Nature.

[39]  R. Cedergren,et al.  The automated chemical synthesis of long oligoribuncleotides using 2'-O-silylated ribonucleoside 3'-O-phosphoramidites on a controlled-pore glass support: synthesis of a 43-nucleotide sequence similar to the 3'-half molecule of an Escherichia coli formylmethionine tRNA , 1987 .

[40]  O. Uhlenbeck.  A small catalytic oligoribonucleotide , 1987, Nature.

[41]  J. Biernat,et al.  Polymer support oligonucleotide synthesis XVIII: use of beta-cyanoethyl-N,N-dialkylamino-/N-morpholino phosphoramidite of deoxynucleosides for the synthesis of DNA fragments simplifying deprotection and isolation of the final product. , 1984, Nucleic acids research.

[42]  Randall R. Sakai,et al.  Mapping of RNA accessible sites for antisense experiments with oligonucleotide libraries , 1998, Nature Biotechnology.

[43]  B. Sproat.  Synthetic Catalytic Oligonucleotides Based on the Hammerhead Ribozyme , 1996 .

[44]  K. Taira,et al.  Mechanistic Studies on Hammerhead Ribozymes , 1996 .

[45]  G. Krupp,et al.  Design of hammerhead ribozymes to distinguish single base changes in substrate RNA. , 1996, Antisense & nucleic acid drug development.

[46]  M. Montenarh,et al.  Antisense effect of oligodeoxynucleotides with inverted terminal internucleotidic linkages: a minimal modification protecting against nucleolytic degradation. , 1992, Antisense research and development.

[47]  I. Tinoco,et al.  Absorbance melting curves of RNA. , 1989, Methods in enzymology.