Structural Rearrangements of the 10–23 DNAzyme to β3 Integrin Subunit mRNA Induced by Cations and Their Relations to the Catalytic Activity

Abstract

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 Mg 2؉ , 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 Mg 2؉ . 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 Mg 2؉ 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 Mg 2؉ may occur in several distinct stages. The first stage, observed at 0.5 mM Mg 2؉ , corresponds to the formation of a compact structure with limited binding properties and without catalytic activity. Then, at 5 mM Mg 2؉ , 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 Mg 2؉ 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.The typical DNAzyme, 1 known as the "10 -23" model, has tremendous potential in gene suppression for both target validation and therapeutic applications (1). It is capable of cleaving single-stranded RNA at specific sites under simulated physiological conditions and can be used to control even complex biological processes such as tumor angiogenesis. For example, DNAzymes to ␤1 and ␤3 mRNA reduced expression of targeted integrin subunits in endothelial cells and blocked proliferation, migration, and network formation in a fibrin and Matrigel™ matrix (2). In a cell culture system, a 10 -23 deoxyribozyme designed against 12-lipoxygenase mRNA specifically down-regulated expression of this protein and its metabolites, which are known to play a crucial role in tumor angiogenesis (3). Similarly, the DNAzyme to VEGFR2 mRNA cleaved its substrate efficiently and inhibited the proliferation of endothelial cells with a concomitant reduction of VEGFR2 mRNA and blocked tumor growth in vivo (4).The origins of the DNAzyme catalytic activity are not yet fully understood, but the observed rate enhancements probably are generated by a number of factors, including metal ion and nucleobase catalysis and local stereochemical effects. The 10 -23 DNAzyme has been developed using an in vitro selection strategy on the basis of it...

Tandem DNAzymes for mRNA cleavage: Choice of enzyme, metal ions and the antisense effect

“…27,28 Biochemical and spectroscopic studies suggest that previously reported 10-23 DNAzyme induced gene silencing might be simply due to an anti-sense effect 4 instead of DNAzyme-induced cleavage. 29,30 Since most of the previous gene silencing work only measured the final mRNA or protein concentration, while the step-by-step reaction processes were not followed, the exact role of the DNAzymes remains elusive. 13 To move forward, a clear understanding on the reaction steps of DNAzyme catalysis is needed.…”

DNAzyme Hybridization, Cleavage, Degradation, and Sensing in Undiluted Human Blood Serum

“…For example, concerns have been raised regarding DNAzyme activity in the presence of physiological concentrations of Mg 2+ . [25][26][27] An important aspect of expanding the analytical application of DNAzymes is to involve biological sample matrices. 28 For example, a common perception is that free DNA might be quickly degraded by nucleases in biological fluids, keeping many researchers away from related studies.…”

Antisense applications for biological control

“…For example, these small nucleic acids do not seem to be structurally robust. One recent study suggested that, under ionic conditions simulating the intracellular milieu, Dzs were only partially folded and showed a largely suboptimal catalytic activity [Cieslak et al, 2003].…”