The active site components and substrate specificity of an RNA Diels-Aldera
se (DA22) were investigated. Diels-Alderase activity was found to be highly
dependent on a unique 5-position pyridyl modified uridine. Even closely re
lated pyridyl modifications failed to yield active catalysts. Substrate spe
cificity of this Diels-Alderase was remarkable. Experiments with alternativ
e diene and dienophile substrates showed the active site of the Diels-Alder
ase to be highly discriminating, even against molecules of similar reactivi
ty and structure. Inhibition studies with a series of product analogues est
ablished that the RNA recognizes functional components in and around the re
action center of both the diene and the dienophile. Taken together, these r
esults suggest that DA22 can fold into a structure that produces an intrica
te metal/ligand dependent active site capable of highly specific molecular
recognition.