RNA-cleaving DNA enzymes with altered regio- or enantioselectivity.
In vitro evolution methods were used to obtain DNA enzymes that cleave either a 2',5'-phosphodiester following a D-ribonucleotide or a 3',5'-phosphodiester following an L-ribonucleotide. Both enzymes can operate in an intermolecular reaction format with multiple turnover. The DNA enzyme that cleaves a 2',5'-phosphodiester exhibits a k(cat) of approximately 0.01 min(-1) and catalytic efficiency, k(cat)/K(m), of approximately 10(8) M(-1) min(-1). The enzyme that cleaves an L-ribonucleotide is about 10-fold slower and has a catalytic efficiency of approximately 4 x 10(5) M(-1) min(-1). Both enzymes require a divalent metal cation for their activity and have optimal catalytic rate at pH 7-8 and 35-50 degrees C. In a comparison of each enzyme's activity with either its corresponding substrate that contains an unnatural ribonucleotide or a substrate that instead contains a standard ribonucleotide, the 2',5'-phosphodiester-cleaving DNA enzyme exhibited a regioselectivity of 6000-fold, while the L-ribonucleotide-cleaving DNA enzyme exhibited an enantioselectivity of 40-fold. These molecules demonstrate how in vitro evolution can be used to obtain regio- and enantioselective catalysts that exhibit specificities for nonnatural analogues of biological compounds.
Ordoukhanian
Phillip
P
Departments of Chemistry, The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Joyce
Gerald F
GF
D021881:DNA, Catalytic / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000737:chemistry; D012313:RNA / Q000737:chemistry* / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity
D021881:DNA, Catalytic; D009841:Oligonucleotides; D012313:RNA