ISOLATION OF A RIBOZYME WITH 5'-5' LIGASE ACTIVITY

Background: Many new ribozymes, including sequence-specific nucleases, ligases and kinases, have been isolated by in vitro selection from la rge pools of random-sequence RNAs. We are attempting to use in vitro s election to isolate new ribozymes that have, or can be evolved to have , RNA polymerase-like activities. As phosphorimidazolide-activated nuc leosides 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 substra te oligonucleotide via their 5'-triphosphate, generating a 5'-5' P-1,P -4-tetraphosphate linkage. Deletion analysis of one of the selected se quences revealed that a 54-nucleotide RNA retained activity; this smal l ribozyme folds into a pseudoknot secondary structure with an interna l binding site for the substrate oligonucleotide. The ribozyme can als o synthesize 5'-5' triphosphate and 5'-5' pyrophosphate linkages. Conc lusions: The emergence of ribozymes that accelerate an unexpected 5'-5 ' ligation reaction from a selection designed to yield template-depend ent 3'-5' ligases suggests that it may be much easier for RNA to catal yze 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 ribozym e-catalyzed synthesis of such linkages raises the possibility that the se 5'-5' linkages originated in the biochemistry of the RNA world.