Background: 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. Results: 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. Conclusions: 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.
Bibliographical noteFunding Information:
This work was supported by the Deutsche Forschungsgemeinschaft (grants # Ja 794/l and SFB 344/BlO to A.J. ). We thank E. Nordhoff for mass spectrometric analysis, M. Ziegler and G. Buchlow for assistance with sequencing and fluorescence spectrometry, and V.A. Erdmann far support. We appreciate helpful suggestions by F. Hausch, C. Frauendorf, and T. Ruppert.
- In vitro selection
- Oligonucleotide conjugates
- RNA catalysis