Structural rearrangements of the 10-23 DNAzyme to beta 3 integrin subunit mRNA induced by cations and their relations to the catalytic activity.
The intracellular ability of the "10-23" DNAzyme to efficiently inhibit expression of targeted proteins has been evidenced by in vitro and in vivo studies. However, standard conditions for kinetic measurements of the DNAzyme catalytic activity in vitro include 25 mM Mg2+, a concentration that is very unlikely to be achieved intracellularly. To study this discrepancy, we analyzed the folding transitions of the 10-23 DNAzyme induced by Mg2+. For this purpose, spectroscopic analyzes such as fluorescence resonance energy transfer, fluorescence anisotropy, circular dichroism, and surface plasmon resonance measurements were performed. The global geometry of the DNAzyme in the absence of added Mg2+ seems to be essentially extended, has no catalytic activity, and shows a very low binding affinity to its RNA substrate. The folding of the DNAzyme induced by binding of Mg2+ may occur in several distinct stages. The first stage, observed at 0.5 mM Mg2+, corresponds to the formation of a compact structure with limited binding properties and without catalytic activity. Then, at 5 mM Mg2+, flanking arms are projected at right position and angles to bind RNA. In such a state, DNAzyme shows substantial binding to its substrate and significant catalytic activity. Finally, the transition occurring at 15 mM Mg2+ leads to the formation of the catalytic domain, and DNAzyme shows high binding affinity toward substrate and efficient catalytic activity. Under conditions simulating intracellular conditions, the DNAzyme was only partially folded, did not bind to its substrate, and showed only residual catalytic activity, suggesting that it may be inactive in the transfected cells and behave like antisense oligodeoxynucleotide.
Cieslak
Marcin
M
Center for Molecular and Macromolecular Studies, Polish Academy of Sciences, 92-215 Lodz, Poland.
Szymanski
Jacek
J
Adamiak
Ryszard W
RW
Cierniewski
Czeslaw S
CS
D016880:Anisotropy; D001483:Base Sequence; D019921:Biotinylation; D002384:Catalysis; D020134:Catalytic Domain; D002412:Cations; D002478:Cells, Cultured; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry; D021881:DNA, Catalytic / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000166:cytology; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D039661:Integrin beta3 / Q000737:chemistry*; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D015636:Magnesium Chloride / Q000494:pharmacology; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000737:chemistry; D017510:Protein Folding; D012313:RNA / Q000737:chemistry; D012333:RNA, Messenger / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D020349:Surface Plasmon Resonance; D014162:Transfection; D014471:Umbilical Veins / Q000166:cytology
D002412:Cations; D021881:DNA, Catalytic; D039661:Integrin beta3; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D015636:Magnesium Chloride; D012313:RNA; D004247:DNA; D008274:Magnesium