A ribozyme and a catalytic DNA with peroxidase activity: active sites versus cofactor-binding sites

Background: An 18-nucleotide DNA oligomer, PS2.M, derived using an in vitro selection method was previously reported to bind hemin (Fe(III)-protoporph yrinIX) with submicromolar affinity. The DNA-hemin complex exhibited DNA-en hanced peroxidative activity. PS2.M is guanine-rich and requires potassium ions to fold to its active conformation, consistent with its forming a guan ine-quaduplex. In investigating the specific catalytic features of PS2.M we tested the peroxidative properties of its RNA version (rPS2.M) as well as that of an unrelated DNA guanine-quadruplex, OXY4. Results: The hemin-binding affinity of rPS2.M was found to be 30-fold weake r than that of PS2.M. The UV-visible spectra and kinetics of enzymatic pero xidation of the RNA-hemin complex, however, were nearly identical to those of its DNA counterpart. Both displayed peroxidase activity substantially gr eater than those of heme proteins such as catalase and Fe(III)-myoglobin. K inetic analysis suggested that PS2.M and rPS2.M catalyzed the breakdown of the hemin-hydrogen peroxide covalent complex to products. The hemin complex of folded OXY4 (which bound hemin as strongly as did rPS2.M) had a distinc t absorption spectrum and only a minor peroxidase activity above the backgr ound level. The results indicated that it is possible for RNA and DNA of the same seque nce to fold to form comparable cofactor-binding sites, and to show comparab le catalytic behavior. The results further suggest that only a subset of co factor-binding sites formed within folded nucleic acids might be able to fu nction as active sites, by providing the appropriate chemical environments for catalysis.