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Phosphorothioate Oligonucleotides Block the VDAC Channel

Wenzhi Tan ^*, Yue-Hin Loke ^*, C. A. Stein , Paul Miller  and Marco Colombini ^*

^* Department of Biology, University of Maryland, College Park, Maryland 20742; Albert Einstein-Montefiore Cancer Center, Department of Oncology, Montefiore Medical Center, Bronx, New York 10467; and The Johns Hopkins University, School of Public Health, Baltimore, Maryland 21287

Correspondence: Address reprint requests to Marco Colombini, Dept. of Biology, University of Maryland, College Park, MD 20742. Tel.: 301 405-6925; Fax: 301 314-9358; E-mail: colombini{at}umd.edu.

Proapoptotic phosphorothioate oligonucleotides such as G3139 (an 18-mer) induce Bcl-2-independent apoptosis, perhaps partly via direct interaction with VDAC and reduction of metabolite flow across the mitochondrial outer membrane. Here, we analyzed the interactions at the molecular level. Ten micromolar G3139 induces rapid flickering of the VDAC conductance and, occasionally, a complete conductance drop. These phenomena occur only when VDAC is in the "open" conformation and therefore are consistent with pore blockage rather than VDAC closure. Blockage occurs preferentially from one side of the VDAC channel. It depends linearly on the [G3139] and is voltage-dependent with an effective valence of –3. The kinetics indicate at least a partial entry of G3139 into VDAC, forming an unstable bound state, which is responsible for the rapid flickering (0.1 ms). Subsequently, a long-lived blocked state is formed. An 8-mer phosphorothioate, polydeoxythymidine, induces partial blockage of VDAC and a change in selectivity from favoring anions to favoring cations. Thus, the oligonucleotide is close to the ion stream. The phosphodiester congener of G3139 is ineffective at the concentrations used, excluding a general polyanion effect. This shows the importance of sulfur atoms. The results are consistent with a binding-induced blockage rather than a permeation block.

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