TOTAL CHEMICAL SYNTHESIS OF A RIBOZYME DERIVED FROM A GROUP-I INTRON

We describe the complete chemical synthesis of a ribozyme that catalyz es template-directed oligonucleotide ligation, The specific activity o f the synthetic ribozyme is nearly identical to that of the same enzym e generated by in vitro transcription with T7 RNA polymerase. The ribo zyme is derived from a group I intron and consists of three RNA fragme nts of 36, 43, and 59 nt that self-assemble to form a catalytically ac tive complex, We have site-specifically substituted ribonucleotide ana logs into this enzyme and have identified two 2'-hydroxyl groups that are required for full catalytic activity, In contrast, neither the 2'- hydroxyl nor the exocyclic amino group of the conserved guanosine in t he guanosine binding site is necessary for catalysis, By allowing the ribozyme to be modified as easily as its substrates, this synthetic ri bozyme system should be useful for testing specific hypotheses concern ing ribozyme-substrate interactions and tertiary interactions within t he ribozyme.