Toward an efficient DNAzyme.
A 33-nucleotide, guanine-rich DNA oligomer, PS5.ST1, has been reported to catalyze the metallation of mesoporphyrin IX (MPIX) by copper and zinc ions. In this paper we report a thorough investigation of the properties of this DNAzyme. We have established that a 24-nucleotide sequence (PS5.M), from within PS5.ST1, is both the minimal and most optimal catalytic unit. We have found that three related porphyrins are acceptable as substrates by this DNAzyme, of which protoporphyrin IX is preferred as a substrate over the expected substrate, MPIX. We have determined that it is unlikely that a strong, catalytically relevant binding site for copper ions exists in the DNAzyme and that high concentrations of copper destroy the active DNAzyme. This enzyme, whose folded structure likely contains guanine quartets, requires potassium ions for activity; we have shown that as little as 1 mM potassium is sufficient for its catalytic robustness, whereas as much as 0.5 M sodium still will not support catalysis. In determining the pH, temperature, and salt optima for the catalyzed reaction, we have found an unexpected stabilizing role for Tris buffer in both the catalyzed and background metallation reactions. As a consequence of various steps of optimization, we now have a vastly improved DNAzyme, one whose enzymatic parameters compare well both with those of natural ferrochelatases, as well as with those of artificially derived chelatases, composed of protein (a catalytic antibody) and RNA. The existence of this array of biocatalysts for porphyrin metallations allows one-to-one comparisons of the ways in which different biopolymers solve a given catalytic problem.
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Institute of Molecular Biology & Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada.
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D001665:Binding Sites; D002021:Buffers; D002384:Catalysis / Q000187:drug effects; D003300:Copper / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D005294:Ferrochelatase / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008652:Mesoporphyrins / Q000378:metabolism; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism*; D011166:Porphyrins / Q000378:metabolism*; D011188:Potassium / Q000494:pharmacology; D011524:Protoporphyrins / Q000378:metabolism; D012964:Sodium / Q000494:pharmacology; D013379:Substrate Specificity
D002021:Buffers; D003902:Detergents; D008652:Mesoporphyrins; D009838:Oligodeoxyribonucleotides; D011166:Porphyrins; D011524:Protoporphyrins; C031468:mesoporphyrin IX; D003300:Copper; D012964:Sodium; D005294:Ferrochelatase; D011188:Potassium; D004121:Dimethyl Sulfoxide