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Biochemical and biophysical study of transition metal ion-dependent DNAzymes
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Journal of Inorganic Biochemistry 96 (2003)
Biochemical and Biophysical Study of Transition Metal Ion-dependent DNAzymes yi University of Illinois at Urbana-Champaign, United States Amdrea K Brown, Department of Chemistry, University of Illinois at Urbana-Champaign, United States Juewen Liu, Department of Chemistry, University of Illinois at Urbana-Champaign, United States Jing Li, Department of Chemistry, University of Illinois at Urbana-Champaign, United States Peter J Bruesehoff Department of Chemistry, University of Illinois at Urbana-Champaign, United States Like proteins and RNA molecules, many DNA molecules have now been shown to catalyze a variety of reactions and are thus called DNAzymes. With limited building blocks, DNAzymes need to recruit cofactors in order to match other enzymes in terms of reaction diversity and catalytic efficiency. Several unique properties make transition metal ions an ideal cofactor for DNAzymes.’ We have obtained new DNAzymes that bind transition metal ions with high affinity and selectivity through the use of a combinatorial biology tool called in vitro selection.2 The work makes 0 it possible to obtain different classesof metallo-DNAzymes in the laboratory within a short period T of time. It also offers a rare opportunity to compare and contrast structural and functional properties of metal-binding sites in proteins and in DNAzymes. A detailed biochemical and mechanistic study3 AA G of a DNAzyme showed that metal-binding affinity of the DNAzymes is in the order of PbZ+> Zr?’ > A Tc MgZ+.While Mgz+ and Zn2’ catalyze only a transesterification reaction with formation of a product containing a 2’,3’-cyclic phosphate, Pb*+catalyzesa transesterification reaction followed by hydrolysis oc of the 2’,3’-cyclic phosphate. Although this two-step mechanism has been shown to be operative in protein ribonucleases and in the leadzyme RNAzyme, it is now demonstrated for the first time that this DNAzyme may also use the same mechanism. Furthermore, we have used FRET to study the transition metal ion-dependent folding of the DNAzymes, and developed a novel strategy of labeling each branch of DNAzyme with a fluorophore SO that the folding branch can be monitored independently.4 We showed that the system is simple and yet powerful in studying complicated biomolecular structure and dynamics, and is capable of revealing new sophisticated structural changes that may have functional implications. I
Lu, Y. Chem. Euro. J. 8,458s (2002); 2. Li, .I. et al., Nucleic Acids Res. 28,481-488 (2000); 3. Brown, A. K. et al., Biochemistry, in press; 4. Liu, J. and Lu, Y., J. Am. Chem. SOC. 122,15208 (2002).
Journal of Inorganic Biochemistry 96 (2003)
Biochemical and Biophysical Study of Transition Metal Ion-dependent DNAzymes yi University of Illinois at Urbana-Champaign, United States Amdrea K Brown, Department of Chemistry, University of Illinois at Urbana-Champaign, United States Juewen Liu, Department of Chemistry, University of Illinois at Urbana-Champaign, United States Jing Li, Department of Chemistry, University of Illinois at Urbana-Champaign, United States Peter J Bruesehoff Department of Chemistry, University of Illinois at Urbana-Champaign, United States Like proteins and RNA molecules, many DNA molecules have now been shown to catalyze a variety of reactions and are thus called DNAzymes. With limited building blocks, DNAzymes need to recruit cofactors in order to match other enzymes in terms of reaction diversity and catalytic efficiency. Several unique properties make transition metal ions an ideal cofactor for DNAzymes.’ We have obtained new DNAzymes that bind transition metal ions with high affinity and selectivity through the use of a combinatorial biology tool called in vitro selection.2 The work makes 0 it possible to obtain different classesof metallo-DNAzymes in the laboratory within a short period T of time. It also offers a rare opportunity to compare and contrast structural and functional properties of metal-binding sites in proteins and in DNAzymes. A detailed biochemical and mechanistic study3 AA G of a DNAzyme showed that metal-binding affinity of the DNAzymes is in the order of PbZ+> Zr?’ > A Tc MgZ+.While Mgz+ and Zn2’ catalyze only a transesterification reaction with formation of a product containing a 2’,3’-cyclic phosphate, Pb*+catalyzesa transesterification reaction followed by hydrolysis oc of the 2’,3’-cyclic phosphate. Although this two-step mechanism has been shown to be operative in protein ribonucleases and in the leadzyme RNAzyme, it is now demonstrated for the first time that this DNAzyme may also use the same mechanism. Furthermore, we have used FRET to study the transition metal ion-dependent folding of the DNAzymes, and developed a novel strategy of labeling each branch of DNAzyme with a fluorophore SO that the folding branch can be monitored independently.4 We showed that the system is simple and yet powerful in studying complicated biomolecular structure and dynamics, and is capable of revealing new sophisticated structural changes that may have functional implications. I
Lu, Y. Chem. Euro. J. 8,458s (2002); 2. Li, .I. et al., Nucleic Acids Res. 28,481-488 (2000); 3. Brown, A. K. et al., Biochemistry, in press; 4. Liu, J. and Lu, Y., J. Am. Chem. SOC. 122,15208 (2002).