Metal-dependent global folding and activity of the 8-17 DNAzyme studied by fluorescence resonance energy transfer

Hee Kyung Kim, Juewen Liu, Jing Li, Nandini Nagraj, Mingxi Li, Caroline M.B. Pavot, Yi Lu

Research output: Contribution to journalArticlepeer-review

Abstract

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 μM 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 Zn 2+ 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+.

Original languageEnglish (US)
Pages (from-to)6896-6902
Number of pages7
JournalJournal of the American Chemical Society
Volume129
Issue number21
DOIs
StatePublished - May 30 2007

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

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