DESIGN & SYNTHESIS OF OLIGONUCLEOTIDE ANALOG ANTIVIRALS

这项研究计划的一个长期目标将是发展 修饰的寡核苷酸，这将抑制人类的能力 巨细胞病毒(HCMV)在人类细胞中感染和复制。最基本的 概念是构建寡核苷酸衍生物，它们是 与病毒必需的病毒mRNA序列互补 复制。寡核苷酸将被设计成通过 催化切割它们的互补序列。三种类型的 将对修改进行调查。其中包括：1)寡核苷酸 稳定的亲脂金属修饰磷酸二酯主链 旨在防止核酸酶裂解和加强运输的复合体； 2)设计用来切割RNA的过渡金属配体络合物 3)氧化还原活性过渡金属络合物 旨在通过激活氧气来裂解DNA和RNA，从而导致 分子中C-L和C-4‘位氢原子的自由基抽提 核糖或脱氧核糖。 在分子模拟的基础上，DG的N-2位修饰 似乎为连接配体提供了最佳位置 位于DNA-DNA或DNA-RNA双螺旋的小凹槽中。 被设计用来通过水解机制作用于RNA的核酸酶将 需要两种类型的修改。N-2脱氧鸟苷将作为 用于将咪唑定位在与2‘-羟基相邻的位置的位置 补充性胞苷。并且，脱氧尿苷的C-5将被 设计用于定位金属络合物以对抗3‘-磷酸的配体 注定要乳沟的。 在研究的初始阶段，将有必要 使用相对简单的模型系统来测试 序列特异性切割信使核糖核酸。将进行模型研究 最初与序列5‘-ATCT*CAGTG*G*TA-3’和 5‘-GAAT*T*CTTTG*CC-3’，与残基489-500和 411-422的兔β-珠蛋白基因。在N-2处引入修改 T*的G*和C-5相隔5个或6个核苷酸单位。 计划进行研究，以确定这些修改如何影响 互补DNA和RNA杂交的特异性和强度 序列。随后添加额外的修饰寡核苷酸 与兔珠蛋白基因中的多个不同区域互补 将进行合成并进行结合和切割实验。 最终，该方法可以适用于控制环境污染。 广泛的病毒感染，包括大多数病毒 经常与艾滋病有关(例如，疱疹病毒、肝炎病毒、 和乳头瘤病毒)。这种方法的模块化性质赋予了它 对广泛的病毒具有潜在的广泛适用性。如果 在此授权期内开发的模型化合物具有以下功能 预计，那么我们将能够设计寡核苷酸 靶向特定的人巨细胞病毒序列。

A long-range goal of this research program will be the development of modified oligonucleotides, which will inhibit the ability of human cytomegalovirus (HCMV) to infect and replicate in human cells. The basic concept is to construct oligonucleotide derivatives, which are complementary to viral mRNA sequences that are essential for viral replication. The oligonucleotides would be designed to function by catalytically cleaving their complementary sequence. Three types of modifications are to be investigated. These include: 1) oligonucleotides modified at the phosphodiester backbone by stable lipophilic metal complexes designed to prevent nuclease cleavage and to enhance transport; 2) transition metal ligand complexes designed to cleave RNA by a hydrolytic mechanism; and 3) redox active transition metal complexes designed to cleave DNA and RNA through activation of dioxygen leading to radical abstraction of hydrogen atoms at the C-l' and C-4'positions of ribose or deoxyribose. On the basis of molecular modeling, modification at N-2 of dG appears to provide an optimal location for attaching ligands that are to be positioned in the minor groove in a DNA-DNA or DNA-RNA double helix. The nucleases designed to operate on RNA by a hydrolytic mechanism will require two types of modification. N-2 of deoxyguanosine will serve as a site for tethering an imidazole to be positioned adjacent to the 2'-OH of the complementary cytidine. And, C-5 of deoxyuridine will be modified by ligands designed to position a metal complex against the 3'-phosphate destined for cleavage. In the initial phases of the research, it will be necessary to use a relatively simple model system in order to test the concept of sequence-specific cleavage of mRNA. Model studies will be carried out initially with the sequences 5'-ATCT*CAGTG*G*TA-3' and 5'-GAAT*T*CTTTG*CC-3', which are complementary to residues 489-500 and 411-422 of rabbit beta-globin mRNA. Modifications are introduced at N-2 of G* and C-5 of T* spaced either five or six nucleotide units apart. Studies are planned to determine how these modifications affect the specificity and strength of hybridization to complementary DNA and RNA sequences. Subsequently additional modified oligonucleotides complementary to a number of different regions in the rabbit globin mRNA will be synthesized and binding and cleavage experiments carried out. Ultimately, the approach could be applicable to the control of a broad range of viral infections, including most of the viruses frequently associated with AIDS (e.g., herpes viruses, hepatitis viruses, and papillomaviruses). The modular nature of the approach gives it potentially broad applicability to a broad range of viruses. If the model compounds developed during this grant period function as anticipated, then we will be in a position to design oligonucleotides targeted to specific HCMV sequences.