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ABSTRACT: The interaction of factor Xa with factor Va on membranes to form prothrombinase profoundly increases the rate of the proteolytic conversion of prothrombin to thrombin. We present the characterization of an RNA aptamer (RNA(11F7t)) selected from a combinatorial library based on its ability to bind factor Xa. We show that RNA(11F7t) inhibits thrombin formation catalyzed by prothrombinase without obscuring the active site of Xa within the enzyme complex. Selective inhibition of protein substrate cleavage arises from the ability of the aptamer to bind to factor Xa and exclude interactions between the proteinase and cofactor within prothrombinase. Competition for enzyme complex assembly results from the binding of RNA(11F7t) to factor Xa with nanomolar affinity in a Ca(2+)-dependent interaction. RNA(11F7t) binds equivalently to the zymogen factor X as well as derivatives lacking gamma-carboxyglutamic acid residues. We suggest that the ability of RNA(11F7t) to compete for the Xa-Va interaction with surprisingly high affinity likely reflects a significant contribution from its ability to indirectly impact regions of Xa that participate in the proteinase-cofactor interaction. Thus, despite the complexity of the macromolecular interactions that underlie the assembly of prothrombinase, efficient inhibition of enzyme complex assembly and thrombin formation can be achieved by tight binding ligands that target factor Xa in a discrete manner.
Journal of Biological Chemistry 12/2009; 285(8):5212-23. · 4.77 Impact Factor
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ABSTRACT: We show that a molecular scaffold can be utilized to convert a receptor binding aptamer into a receptor agonist. Many receptors (including tumor necrosis receptor family members) are activated when they are multimerized on the cell surface. Molecular scaffolds have been utilized to assemble multiple receptor binding peptide ligands to generate activators of such receptors. We demonstrate that an RNA aptamer that recognizes OX40, a member of the tumor necrosis factor receptor superfamily, can be converted into a receptor-activating aptamer by assembling two copies on an olignucleotide-based scaffold. The OX40 receptor-activating aptamer is able to induce nuclear localization of nuclear factor-kappaB, cytokine production, and cell proliferation, as well as enhance the potency of dendritic cell-based tumor vaccines when systemically delivered to mice.
Chemistry & Biology 07/2008; 15(7):675-82. · 5.83 Impact Factor
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Paloma H Giangrande,
JianXin Zhang,
Alice Tanner,
Andrea D Eckhart,
Rachel E Rempel,
Eran R Andrechek, Juliana M Layzer,
Janelle R Keys,
Per-Otto Hagen,
Joseph R Nevins,
Walter J Koch,
Bruce A Sullenger
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ABSTRACT: Intimal hyperplasia (IH) and restenosis limit the long-term utility of bypass surgery and angioplasty due to pathological proliferation and migration of vascular smooth muscle cells (VSMCs) into the intima of treated vessels. Consequently, much attention has been focused on developing inhibitory agents that reduce this pathogenic process. The E2F transcription factors are key cell cycle regulators that play important roles in modulating cell proliferation and cell fate. Nonselective E2F inhibitors have thus been extensively evaluated for this purpose. Surprisingly, these E2F inhibitors have failed to reduce IH. These findings prompted us to evaluate the roles of different E2Fs during IH to determine how selective targeting of E2F isoforms impacts VSMC proliferation. Importantly, we show that E2F3 promotes proliferation of VSMCs leading to increased IH, whereas E2F4 inhibits this pathological response. Furthermore, we use RNA probes to show that selective inhibition of E2F3, not global inhibition of E2F activity, significantly reduces VSMC proliferation and limits IH in murine bypass grafts.
Proceedings of the National Academy of Sciences 09/2007; 104(32):12988-93. · 9.68 Impact Factor
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ABSTRACT: By using the in vitro selection method SELEX against the complex mixture of GLA proteins and utilizing methods to deconvolute the resulting ligands, we were able to successfully generate 2'-ribo purine, 2'-fluoro pyrimidine aptamers to various individual targets in the GLA protein proteome that ranged in concentration from 10 nM to 1.4 microM in plasma. Perhaps not unexpectedly, the majority of the aptamers isolated following SELEX bind the most abundant protein in the mixture, prothrombin (FII), with high affinity. We show that by deselecting the dominant prothrombin aptamer the selection can be redirected. By using this DeSELEX approach, we were able to shift the selection toward other sequences and to less abundant protein targets and obtained an aptamer to Factor IX (FIX). We also demonstrate that by using an RNA library that is focused around a proteome, purified protein targets can then be used to rapidly generate aptamers to the protein targets that are rare in the initial mixture such as Factor VII (FVII) and Factor X (FX). Moreover, for all four proteins targeted (FII, FVII, FIX, and FX), aptamers were identified that could inhibit the individual protein's activitity in coagulation assays. Thus, by applying the concepts of DeSELEX and focused library selection, aptamers specific for any protein in a particular proteome can theoretically be generated, even when the proteins in the mixture are present at very different concentrations.
Oligonucleotides 02/2007; 17(1):1-11. · 2.80 Impact Factor
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ABSTRACT: Chemical modifications have been incorporated into short interfering RNAs (siRNAs) without reducing their ability to inhibit gene expression in mammalian cells grown in vitro. In this study, we begin to assess the potential utility of 2'-modified siRNAs in mammals. We demonstrate that siRNA modified with 2'-fluoro (2'-F) pyrimidines are functional in cell culture and have a greatly increased stability and a prolonged half-life in human plasma as compared to 2'-OH containing siRNAs. Moreover, we show that the 2'-F containing siRNAs are functional in mice and can inhibit the expression of a target gene in vivo. However, even though the modified siRNAs have greatly increased resistance to nuclease degradation in plasma, this increase in stability did not translate into enhanced or prolonged inhibitory activity of target gene reduction in mice following tail vein injection. Thus, this study shows that 2'-F modified siRNAs are functional in vivo, but that they are not necessarily more potent than unmodified siRNAs in animals.
RNA 06/2004; 10(5):766-71. · 5.09 Impact Factor
