title: Isolation of a ribozyme with 5'-5' ligase activity.
abstract: BACKGROUND
  Many new ribozymes, including sequence-specific nucleases, ligases and kinases, have been isolated by in vitro selection from large pools of random-sequence RNAs. We are attempting to use in vitro selection to isolate new ribozymes that have, or can be evolved to have, RNA polymerase-like activities. As phosphorimidazolide-activated nucleosides are extensively used to study non-enzymatic RNA replication, we wished to select for a ribozyme that would accelerate the template-directed ligation of 5'-phosphorimidazolide-activated oligonucleotides.
  
  
  RESULTS
  Ribozymes selected to perform the desired template-directed ligation reaction instead ligated themselves to the activated substrate oligonucleotide via their 5'-triphosphate, generating a 5'-5' P1,P4-tetraphosphate linkage. Deletion analysis of one of the selected sequences revealed that a 54-nucleotide RNA retained activity; this small ribozyme folds into a pseudoknot secondary structure with an internal binding site for the substrate oligonucleotide. The ribozyme can also synthesize 5'-5' triphosphate and 5'-5' pyrophosphate linkages.
  
  
  CONCLUSIONS
  The emergence of ribozymes that accelerate an unexpected 5'-5' ligation reaction from a selection designed to yield template-dependent 3'-5' ligases suggests that it may be much easier for RNA to catalyze the synthesis of 5'-5' linkages than 3'-5' linkages. 5'-5' linkages are found in a variety of contexts in present-day biology. The ribozyme-catalyzed synthesis of such linkages raises the possibility that these 5'-5' linkages originated in the biochemistry of the RNA world.
authors.affiliation: Department of Molecular Biology, Massachusetts General Hospital, Boston 02114, USA.
authors.forename: K B
authors.initials: KB
authors.lastname: Chapman
authors.forename: J W
authors.initials: JW
authors.lastname: Szostak
mesh_terms: D001483:Base Sequence; D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D019020:Directed Molecular Evolution; D008025:Ligases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism; D016337:RNA, Catalytic / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism
chemical_list: D009841:Oligonucleotides; D016337:RNA, Catalytic; D012321:DNA-Directed RNA Polymerases; D015719:Single-Strand Specific DNA and RNA Endonucleases; D008025:Ligases