ISOLATION OF NOVEL RIBOZYMES THAT LIGATE AMP-ACTIVATED RNA SUBSTRATES

Background: The protein enzymes RNA ligase and DNA ligase catalyze the ligation of nucleic acids via an adenosine-5'-5'-pyrophosphate 'cappe d' RNA or DNA intermediate, The activation of nucleic acid substrates by adenosine 5'-monophosphate (AMP) may be a vestige of 'RNA world' ca talysis. AMP-activated ligation seems ideally suited for catalysis by ribozymes (RNA enzymes), because an RNA motif capable of tightly and s pecifically binding AMP has previously been isolated. Results: We used in vitro selection and directed evolution to explore the ability of r ibozymes to catalyze the template-directed ligation of AMP-activated R NAs. We subjected a pool of 10(15) RNA molecules, each consisting of l ong random sequences flanking a mutagenized adenosine triphosphate (AT P) aptamer, to ten rounds of in vitro selection, including three round s involving mutagenic polymerase chain reaction, Selection was for the ligation of an oligonucleotide to the 5'-capped active pool RNA speci es. Many different ligase ribozymes were isolated; these ribozymes had rates of reaction up to 0.4 ligations per hour, corresponding to rate accelerations of similar to 5 x 10(5) over the templated, but otherwi se uncatalyzed, background reaction rate, Three characterized ribozyme s catalyzed the formation of 3'-5'-phosphodiester bonds and were highl y specific for activation by AMP at the ligation site. Conclusions: Th e existence of a new class of ligase ribozymes is consistent with the hypothesis that the unusual mechanism of the biological ligases result ed from a conservation of mechanism during an evolutionary replacement of a primordial ribozyme ligase by a more modern protein enzyme. The newly isolated ligase ribozymes may also provide a starting point for the isolation of ribozymes that catalyze the polymerization of AMP-act ivated oligonucleotides or mononucleotides, which might have been the prebiotic analogs of nucleoside triphosphates.