Directed evolution of an RNA enzyme.

An in vitro evolution procedure was used to obtain RNA enzymes with a particular catalytic function. A population of 10(13) variants of the Tetrahymena ribozyme, a group I ribozyme that catalyzes sequence-specific cleavage of RNA via a phosphoester transfer mechanism, was generated. This enzyme has a limited ability to cleave DNA under conditions of high temperature or high MgCl2 concentration, or both. A selection constraint was imposed on the population of ribozyme variants such that only those individuals that carried out DNA cleavage under physiologic conditions were amplified to produce "progeny" ribozymes. Mutations were introduced during amplification to maintain heterogeneity in the population. This process was repeated for ten successive generations, resulting in enhanced (100 times) DNA cleavage activity.

[1]  G. S. Eadie.  THE INHIBITION OF CHOLINESTERASE BY PHYSOSTIGMINE AND PROSTIGMINE , 1942 .

[2]  B. Hofstee,et al.  Specificity of esterases. I. Identification of two pancreatic aliesterases. , 1952, The Journal of biological chemistry.

[3]  D. Mills,et al.  An extracellular Darwinian experiment with a self-duplicating nucleic acid molecule. , 1967, Proceedings of the National Academy of Sciences of the United States of America.

[4]  D. Hogness,et al.  Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. , 1975, Proceedings of the National Academy of Sciences of the United States of America.

[5]  F. Sanger,et al.  DNA sequencing with chain-terminating inhibitors. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[6]  R. Dixon,et al.  Hydroxylamine mutagenesis of HSV DNA and DNA fragments: introduction of mutations into selected regions of the viral genome. , 1979, Virology.

[7]  D. Holmes,et al.  A rapid boiling method for the preparation of bacterial plasmids. , 1981, Analytical biochemistry.

[8]  L. Loeb,et al.  Site-specific mutagenesis by error-directed DNA synthesis , 1982, Nature.

[9]  T. Cech,et al.  Self-splicing RNA: Autoexcision and autocyclization of the ribosomal RNA intervening sequence of tetrahymena , 1982, Cell.

[10]  M. Chamberlin,et al.  4 Bacteriophage DNA-Dependent RNA Polymerases , 1982 .

[11]  R. Waring,et al.  Making ends meet: a model for RNA splicing in fungal mitochondria , 1982, Nature.

[12]  D. Botstein,et al.  Directed mutagenesis with sodium bisulfite. , 1983, Methods in enzymology.

[13]  H. Heyneker,et al.  Targeted random mutagenesis: the use of ambiguously synthesized oligonucleotides to mutagenize sequences immediately 5' of an ATG initiation codon. , 1983, Nucleic acids research.

[14]  S. Inouye,et al.  Improvement Of Oligonucleotide-Directed Site-Specific Mutagenesis Using Double-Stranded Plasmid DNA , 1984, Bio/Technology.

[15]  M. Vasser,et al.  Cassette mutagenesis: an efficient method for generation of multiple mutations at defined sites. , 1985, Gene.

[16]  Sequence requirements for self-splicing of the Tetrahymena thermophila pre-ribosomal RNA. , 1985, Nucleic acids research.

[17]  R. Myers,et al.  A general method for saturation mutagenesis of cloned DNA fragments. , 1985, Science.

[18]  R. Zagursky,et al.  Rapid and easy sequencing of large linear double-stranded DNA and supercoiled plasmid DNA , 1985 .

[19]  K. Mullis,et al.  Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. , 1985, Science.

[20]  T. Cech,et al.  New reactions of the ribosomal RNA precursor of Tetrahymena and the mechanism of self-splicing. , 1986, Journal of molecular biology.

[21]  J. Szostak.  Enzymatic activity of the conserved core of a group I self-splicing intron , 1986, Nature.

[22]  T. Cech,et al.  The intervening sequence RNA of Tetrahymena is an enzyme. , 1986, Science.

[23]  P. Carter,et al.  Site-directed mutagenesis. , 1986, The Biochemical journal.

[24]  T. Cech,et al.  The Tetrahymena ribozyme acts like an RNA restriction endonuclease , 1986, Nature.

[25]  C. Hutchison,et al.  A complete library of point substitution mutations in the glucocorticoid response element of mouse mammary tumor virus. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[26]  J. J. Salvo,et al.  A simple and efficient procedure for saturation mutagenesis using mixed oligodeoxynucleotides. , 1986, Gene.

[27]  O. Uhlenbeck,et al.  Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates. , 1987, Nucleic acids research.

[28]  G. F. Joyce,et al.  Structure of the catalytic core of the Tetrahymena ribozyme as indicated by reactive abbreviated forms of the molecule. , 1987, Nucleic acids research.

[29]  Tan Inoue,et al.  Catalysis of splicing-related reactions between dinucleotides by a ribozyme , 1987, Nature.

[30]  Sung-Hou Kim,et al.  Three-dimensional model of the active site of the self-splicing rRNA precursor of Tetrahymena. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[31]  H. Tabak,et al.  Structural conventions for group I introns. , 1987, Nucleic acids research.

[32]  J. Knowles.  Tinkering with enzymes: what are we learning? , 1987, Science.

[33]  M. Eigen,et al.  Molecular quasi-species. , 1988 .

[34]  A new method for random mutagenesis of complete genes: enzymatic generation of mutant libraries in vitro. , 1988, Protein engineering.

[35]  T. Cech,et al.  Deletion of nonconserved helices near the 3' end of the rRNA intron of Tetrahymena thermophila alters self-splicing but not core catalytic activity. , 1988, Genes & development.

[36]  T. Cech,et al.  Sequence-specific endoribonuclease activity of the Tetrahymena ribozyme: enhanced cleavage of certain oligonucleotide substrates that form mismatched ribozyme-substrate complexes. , 1988, Biochemistry.

[37]  T. Cech,et al.  Conserved sequences and structures of group I introns: building an active site for RNA catalysis--a review. , 1988, Gene.

[38]  T. Cech,et al.  RNA as an RNA polymerase: net elongation of an RNA primer catalyzed by the Tetrahymena ribozyme. , 1988, Science.

[39]  K. Mullis,et al.  Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. , 1988, Science.

[40]  T. Cech,et al.  Reverse self-splicing of the tetrahymena group I intron: Implication for the directionality of splicing and for intron transposition , 1989, Cell.

[41]  G. F. Joyce,et al.  Catalytic activity is retained in the Tetrahymena group I intron despite removal of the large extension of element P5. , 1989, Nucleic acids research.

[42]  T. Cech,et al.  Alteration of substrate specificity for the endoribonucleolytic cleavage of RNA by the Tetrahymena ribozyme. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[43]  François Michel,et al.  The guanosine binding site of the Tetrahymena ribozyme , 1989, Nature.

[44]  Jennifer A. Doudna,et al.  RNA-catalysed synthesis of complementary-strand RNA , 1989, Nature.

[45]  T. Gingeras,et al.  Transcription-based amplification system and detection of amplified human immunodeficiency virus type 1 with a bead-based sandwich hybridization format. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[46]  G. F. Joyce,et al.  Amplification, mutation and selection of catalytic RNA. , 1989, Gene.

[47]  J. Scott,et al.  Searching for peptide ligands with an epitope library. , 1990, Science.

[48]  L. Gold,et al.  Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. , 1990, Science.

[49]  Gerald F. Joyce,et al.  Selection in vitro of an RNA enzyme that specifically cleaves single-stranded DNA , 1990, Nature.

[50]  R. Green,et al.  Mutational analysis of conserved nucleotides in a self-splicing group I intron. , 1990, Journal of molecular biology.

[51]  R. Green,et al.  In vitro genetic analysis of the Tetrahymena self-splicing intron , 1990, Nature.

[52]  D. Richman,et al.  Isothermal, in vitro amplification of nucleic acids by a multienzyme reaction modeled after retroviral replication. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[53]  M. Rossmann,et al.  Preliminary investigation of the phage φX174 crystal structure , 1990 .

[54]  G. F. Joyce,et al.  Minimum secondary structure requirements for catalytic activity of a self-splicing group I intron. , 1990, Biochemistry.

[55]  E. Westhof,et al.  Modelling of the three-dimensional architecture of group I catalytic introns based on comparative sequence analysis. , 1990, Journal of molecular biology.

[56]  J. Szostak,et al.  In vitro selection of RNA molecules that bind specific ligands , 1990, Nature.

[57]  R A Kerr,et al.  NASA's Search for ETs Hits a Snag on Earth: The space agency has a new device to listen for signals from the cosmos but it is hearing derision from Congress. , 1990, Science.

[58]  Daniel Herschlag,et al.  DNA cleavage catalysed by the ribozyme from Tetrahymena , 1990, Nature.

[59]  R. Barrett,et al.  Peptides on phage: a vast library of peptides for identifying ligands. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[60]  F. Michel,et al.  Mechanism of 3' splice site selection by the catalytic core of the sunY intron of bacteriophage T4: the role of a novel base-pairing interaction in group I introns. , 1990, Genes & development.

[61]  T. Cech,et al.  Catalysis of RNA cleavage by the Tetrahymena thermophila ribozyme. 1. Kinetic description of the reaction of an RNA substrate complementary to the active site. , 1990, Biochemistry.

[62]  X P Zhang,et al.  Random mutagenesis of gene-sized DNA molecules by use of PCR with Taq DNA polymerase. , 1991, Nucleic acids research.

[63]  John M. Burke,et al.  Novel guanosine requirement for catalysis by the hairpin ribozyme , 1991, Nature.

[64]  T. Cech,et al.  Mutations in a nonconserved sequence of the Tetrahymena ribozyme increase activity and specificity , 1991, Cell.

[65]  K. Lam,et al.  A new type of synthetic peptide library for identifying ligand-binding activity , 1992, Nature.

[66]  T. Cech,et al.  Visualizing the higher order folding of a catalytic RNA molecule. , 1991, Science.

[67]  G. F. Joyce,et al.  Randomization of genes by PCR mutagenesis. , 1992, PCR methods and applications.