RNA ENZYMES WITH 2 SMALL-MOLECULE SUBSTRATES

Background: The 'RNA world' hypothesis posits ancient organisms employ ing versatile catalysis by RNAs. In particular, such a metabolism woul d have required RNA catalysts that join small molecules. Such anabolic reactions now occur very widely, for example in phospholipid, terpene , amino acid and nucleotide synthetic pathways in modern organisms. Pr esent RNA systems, however, do not perform such reactions using substr ates that do not base pair. Here we ask whether this lack is a methodo logical artifact due to the practice of selection-amplification, or a fundamental property of active sites reconstructed within RNA structur es. Results: Three rationally modified RNA enzymes, Iso6-G, Iso6-2G an d Iso6-3G, catalyze the formation of (5'-->5') polyphosphate-linked ol igonucleotides in trans. One of these, Iso6-G RNA, has a specific subs trate site for a guanosine triphosphate, GTP, dGTP or ddGTP, and one n onspecific substrate site for a terminal-phosphate-containing small mo lecule. This ribozyme catalyzes multiple turnovers, proceeding at a co nstant rate. Guanosine specificity is probably not attributable to Wat son-Crick base pairing. Conclusions: Ribozymes can readily bind multip le small-molecule substrates simultaneously and catalyze reactions tha t build up larger products, apparently independent of substrate-RNA Wa tson-Crick base pairing. RNA enzymes therefore parallel proteins, whic h often overcome the entropic difficulties of positioning multiple sma ll substrates for catalysis of anabolic reactions. These results suppo rt the idea of a complex ancestral metabolism based on RNA catalysis.