COMBINATORIAL & RATIONAL DESIGN APTAMERS TARGETING HIV

新的组合和基于结构的设计方法将被用来开发硫代和硫代磷酸DNA诱骗或适配子作为针对人类免疫缺陷病毒(HIV)的靶向治疗药物。这些抗艾滋病药物的开发将通过核磁共振波谱和试剂和蛋白质试剂复合体的计算生物化学来促进。我们将专门合成针对HIV-1逆转录酶(RT)和核衣壳(NCp7)以及人转录因子NF-kappaB的硫化骨架适配子寡核苷酸类似物。我们最近开发了一种新的针对人白介素6核因子(NF-IL6)的硫代杂交骨架适配子的组合选择方案，这种转录因子参与诱导急性时相反应和炎症反应中的细胞因子基因启动子。利用随机组合选择的方法和dNTP(α)S S进行聚合酶链式反应扩增，我们选择了与核因子-IL-6结合的特异性最高的硫代取代物(NM范围)。这一点目前正在扩展到核因子-kappaB，也将适用于NCp7和RT。将开发一种分裂合成方案来组合选择这些蛋白质的二硫代磷酸适体。由于硫代和二硫代磷酸取代的寡核苷酸显示核酸酶活性降低，这些组合硫代磷酸选择实验可以为快速鉴定新的治疗药物提供广泛的应用。这项技术将使我们能够开发原则上针对5种不同形式的核因子-kappaB/Rel的5种同源和异源二聚体的15种可能组合中的任何一种的单独适配子。核因子-kappaB/Rel转录因子是免疫和急性期反应、细胞凋亡、细胞增殖和分化的关键介质，也是作用于HIV-1 LTR的关键反式激活因子。因此，它们是控制HIV-1增殖的潜在治疗目标。核磁共振将被用来确定单硫代和二硫代磷酸修饰的寡核苷酸试剂和适体NCp7复合体的三维结构。我们还将评估适体在组织培养测试中的活性，以阻止HIV-1的增殖和基因表达，以及激活HIV基因表达，以确定潜在的感染库。最后，我们将探索利用这些高度选择性的硫代核酸适体识别蛋白质相互作用的可行性，使用一种新的DNA/蛋白质芯片技术在功能蛋白质表达水平上进行遗传分析。

Novel combinatorial and structure-based design methods will be used to develop phosphorothioate and phosphorodithioate DNA decoys or aptamers as targeted therapeutics towards the human immunodeficiency virus (HIV). Development of these anti-AIDS agents will be facilitated by nuclear magnetic resonance (NMR) spectroscopy and computational biochemistry of both agent and protein agent complexes. We will specifically synthesize thioated backbone aptamer oligonucleotide analogues targeted to HIV-1 reverse transcriptase (RT) and nucleocapsid (NCp7) and the human transcription factor NF-kappaB. We have recently developed a novel combinatorial selection scheme for phosphorothioate hybrid backbone aptamers targeting the nuclear factor for human IL6 (NF-IL6), a transcription factor involved in the induction of acute-phase responsive and cytokine gene promoters in response to inflammation. Using a random combinatorial selection approach and dNTP(alpha)S s in PCR amplification, we have selected specific thio-substituted agents which have the highest specificity in binding (nM range) to NF-IL6. This is currently being extended to NF-kappaB and will also be applied to NCp7 and RT. A split synthesis scheme will be developed for combinatorial selection of dithiophosphate aptamers for these proteins. Since phosphorothioate and phosphorodithioate substituted oligonucleotides show reduced nuclease activity, these combinatorial thiophosphate-selection experiments can offer wide application for rapid identification of new therapeutic agents. This technology will allow us to develop separate aptamers targeting in principle any one of the 15 possible combinations of 5 homo- and heterodimers of the 5 different forms of NF-kappaB/Rel. NF-kappaB/Rel transcription factors, are key mediators of the immune and acute phase responses, apoptosis, cell proliferation and differentiation, and are key transactivators acting on the LTR of HIV-1. They thus represent potential therapeutic targets for control of HIV-1 proliferation. NMR will be used to define the three-dimensional structure of monothio- and dithiophosphate modified oligonucleotide agents and aptamer NCp7 complexes. We will also assess the in vivo activity of the aptamers in tissue culture testing to arrest HIV-1 proliferation and gene expression as well as to activate HIV gene expression to identify hidden reservoirs of infection. Finally, we will explore the feasibility of utilizing these highly selective thioaptamers for recognition of protein-protein interactions using a new DNA/protein chip technology for genetic analysis at the level of functional protein expression.