Isolation of a ribozyme with 5′-5′ ligase activity Original Research Article

Isolation of a ribozyme with 5′-5′ ligase activity Original Research Article Pages 325-333 Karen B. Chapman, Jack W. Szostak Close preview | PDF (1584 K) | Related articles | Related reference work articles AbstractAbstract | ReferencesReferences 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.