COMBINATORIAL & RATIONAL DESIGN APTAMERS TARGETING HIV

新型组合和基于结构的设计方法将用于开发硫代磷酸和二硫代磷酸DNA诱饵或适体，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（NMR）光谱和计算生物化学试剂和蛋白质试剂复合物来促进。 我们将专门合成针对HIV-1逆转录酶（RT）和核衣壳（NCp 7）以及人转录因子NF-κ B的硫代骨架适体寡核苷酸类似物。 我们最近开发了一种新的组合选择方案的硫代磷酸酯杂合骨架适体靶向核因子的人IL 6（NF-IL 6），参与诱导急性期响应和细胞因子基因启动子响应炎症的转录因子。 在PCR扩增中使用随机组合选择方法和dNTP（α）S s，我们选择了在与NF-IL 6的结合（nM范围）中具有最高特异性的特定硫代取代试剂。 这是目前正在扩展到NF-κ B，也将适用于NCp 7和RT。分裂合成方案将开发这些蛋白质的二硫代磷酸适配体的组合选择。 由于硫代磷酸酯和二硫代磷酸酯取代的寡核苷酸显示降低的核酸酶活性，这些组合的硫代磷酸酯选择实验可以提供广泛的应用，用于快速鉴定新的治疗剂。 该技术将允许我们开发单独的适配体，其原则上靶向5种不同形式的NF-κ B/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。 NF-κ B/Rel转录因子是免疫和急性期应答、细胞凋亡、细胞增殖和分化的关键介质，并且是作用于HIV-1的LTR的关键反式激活因子。 因此，它们代表了控制HIV-1增殖的潜在治疗靶点。NMR将用于定义单硫代和二硫代磷酸修饰的寡核苷酸试剂和适体NCp 7复合物的三维结构。 我们还将评估适体在组织培养测试中的体内活性，以阻止HIV-1增殖和基因表达，以及激活HIV基因表达以识别隐藏的感染宿主。 最后，我们将探索利用这些高选择性的硫代适体识别蛋白质-蛋白质相互作用的可行性，使用新的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.