A small catalytic RNA motif with Diels-Alderase activity

Abstract

The 'RNA world' hypothesis requires that RNA be able to catalyze a wide variety of chemical reactions. In vitro selection from combinatorial RNA libraries has been used to identify several catalytic activities, most of which have resulted in a self-modification of RNA at one of its constituents. The formation of carbon-carbon bonds is considered an essential prerequisite for a complex metabolism based on RNA. We describe the selection and characterization of new ribozymes that catalyze carbon-carbon bond formation by Diels-Alder reaction of a biotinylated maleimide with an RNA-tethered anthracene. Secondary structure analysis identified a 49-nucleotide RNA motif that accelerates the reaction about 20,000-fold. The motif has only 11 conserved nucleotides that are present in most of the selected sequences. The ribozyme motif is remarkably adaptable with respect to cofactor and metal-ion requirements. The motif was also re-engineered to give a 38-mer RNA that can act as a 'true' catalyst on short external substrate oligonucleotide-anthracene conjugates. We have identified a small, highly abundant RNA motif that can solve the complex task of forming two carbon-carbon bonds between two reactants in trans, a catalytic capacity useful for creating prebiotically relevant molecules. This is the smallest and fastest RNA catalyst for carbon-carbon bond formation reported to date.