Metal-dependent global folding and activity of the 8-17 DNAzyme studied by fluorescence resonance energy transfer.
The 8-17 DNAzyme is a DNA metalloenzyme catalyzing RNA transesterification in the presence of divalent metal ions, with activity following the order Pb2+ >> Zn2+ >>Mg2+. Since the DNAzyme has been used as a metal ion sensor, its metal-induced global folding was studied by fluorescence resonance energy transfer (FRET) by labeling the three stems of the DNAzyme with the Cy3/Cy5 FRET pair two stems at a time in order to gain deeper insight into the role of different metal ions in its structure and function. FRET results indicated that, in the presence of Zn2+ and Mg2+, the DNAzyme folds into a compact structure, stem III approaching a configuration defined by stems I and II without changing the angle between stems I and II. Correlations between metal-induced folding and activity were also studied. For Zn2+ and Mg2+, the metal ion with higher affinity for the DNAzyme in global folding (Kd(Zn) = 52.6 microM and Kd(Mg) = 1.36 mM) also displays higher affinity in activity (Kd(Zn) = 1.15 mM and Kd(Mg) = 53 mM) under the same conditions. Global folding was saturated at much lower concentrations of Zn2+ and Mg2+ than the cleavage activities, indicating the global folding of the DNAzyme occurs before the cleavage activity for those metal ions. Surprisingly, no Pb2+-dependent global folding was observed. These results suggest that for Pb2+ global folding of the DNAzyme may not be a necessary step in its function, which may contribute to the DNAzyme having the highest activity in the presence of Pb2+.
Kim
Hee-Kyung
HK
Department of Chemistry and Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
Liu
Juewen
J
Li
Jing
J
Nagraj
Nandini
N
Li
Mingxi
M
Pavot
Caroline M-B
CM
Lu
Yi
Y
D001483:Base Sequence; D021881:DNA, Catalytic / Q000737:chemistry / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D007854:Lead / Q000737:chemistry*; D008274:Magnesium / Q000737:chemistry*; D009690:Nucleic Acid Conformation; D015032:Zinc / Q000737:chemistry*
D021881:DNA, Catalytic; C407482:RNA-cleaving DNA 8-17; D007854:Lead; D008274:Magnesium; D015032:Zinc