MODIFIELD OLIGONUCLEOTIDES IN TRIPLE HELIX FORMATION

这项研究的主要目标是化学精炼一类序列- 特异性DNA结合三螺旋形成寡核苷酸（TFO）。 的 这些TFO的独特特征是第三链与其 双链体DNA靶标不依赖于pH，并通过GGC和达特稳定 三联体，使得优选取向将结合的TFO 反平行于下面的嘌呤链 复式。 我们最初的研究是用38个碱基长的寡核苷酸（HIV 38 p）， 被设计为与长末端重复序列中的启动子区结合 人类免疫缺陷病毒（HIV）的序列，表明HIV 38 p具有 由于高G含量而倾向于自缔合。 随后的研究 用HIV 38 p进行的研究表明，用2 ′-脱氧-7- 脱氮鸟苷残基显著降低了自缔合。 因此，在本发明中， HIV 38 p中的一些或全部鸟嘌呤残基被选择的 鸟嘌呤类似物可以减少或消除自缔合并改善 三链体形成 我们现在建议制备含有各种取代水平的TFO 范围为10 - 50%，用2 ′-脱氧-7-脱氮鸟苷或2 ′-脱氧-6- 使用固相亚磷酰胺化学的硫代鸟苷。 的 由此获得的修饰的寡核苷酸将被纯化， 表征了 这些新型TFO形成稳定三链体的能力 将使用带移分析、DNA酶足迹法 实验、解链曲线和CD测量。 希望这样的 取代改善了三螺旋结构，减少了自缔合， 增加结合亲和力和抗病毒（HIV和HSV）活性。 详细 第二阶段将评估这种改良的TFO的生物效应 问题研究

The primary goal of this study is to chemically refine a class of sequence- specific, DNA binding, triple helix-forming oligonucleotides (TFOs). the unique feature of these TFOs is that binding of the third strand to its duplex DNA target is pH independent and is stabilized by GGC and TAT triplets such that the preferred orientation places the bound TFO antiparallel with respect to more purine rich strand of the underlying duplex. Our initial studies with a 38 base long oligonucleotide (HIV38p), designed to bind to the promoter region in the long terminal repeat sequence of human immunodeficiency virus (HIV), indicated that HIV38p has a tendency to self-associate due to high G content. Subsequent studies with HIV38p have shown that substitution of G with 2'-deoxy-7- deazaguanosine residues reduces the self-association considerably. Thus, substitution of some or all guanine residues in HIV38p with selected guanine analogs may reduce or eliminate self-association and improve triplex formation. We now propose to prepare TFOs containing various levels of substitutions ranging from 10 to 50% with 2'-deoxy-7-deazaguanosine or 2'-deoxy-6- thioguanosine employing solid-phase phosphoramidite chemistry. The modified oligonucleotides thus obtained will be purified and adequately characterized. The ability of these novel TFOs to form stable triplexes will be evaluated using band shift analysis, DNase footprinting experiments, melting curves and CD measurements. It is hoped that such a substitution improves triple helix structure, reduces self-association, increases binding affinity and antiviral (HIV and HSV) activity. Detailed biological effects of such modified TFOs will be assessed in Phase II studies.