DESIGN & SYNTHESIS OF OLIGONUCLEOTIDE ANALOG ANTIVIRALS

这项研究计划的一个长期目标是开发 修饰的寡核苷酸，这将抑制人类的能力， 巨细胞病毒（HCMV）感染并在人细胞中复制。 基本 概念是构建寡核苷酸衍生物，其 与病毒mRNA序列互补，所述病毒mRNA序列是病毒 复制的 这些寡核苷酸的设计目的是通过 催化裂解它们的互补序列。 三种类型的 修改将被调查。 这些包括：1）寡核苷酸 在磷酸二酯骨架上被稳定的亲脂性金属修饰 设计成防止核酸酶切割和增强转运的复合物; 2)过渡金属配体络合物，其被设计成通过 水解机理; 3）氧化还原活性过渡金属配合物 设计用于通过激活分子氧切割DNA和RNA， 在C-I '和C-4'位置的氢原子的自由基夺取 核糖或脱氧核糖。 在分子模拟的基础上，对dG的N-2位进行了修饰， 似乎提供了连接配体的最佳位置， 位于DNA-DNA或DNA-RNA双螺旋的小沟中。 设计成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脱氧鸟苷将作为一种 用于束缚咪唑的位点位于与2 '-OH相邻的位置 互补胞苷。 脱氧尿苷的C-5将被修饰， 配体被设计为将金属络合物定位在3'-磷酸盐上 注定要乳沟。 在研究的最初阶段，必须 使用一个相对简单的模型系统，以测试的概念 mRNA的序列特异性切割。 将进行模型研究 最初具有序列5'-ATCT * CAGTG * G * TA-3 '， 5'-GAAT * T * CTTTG * CC-3 '，其与残基489 - 500互补，以及 411 - 422的兔β-珠蛋白mRNA。 在N-2处引入修改 G * 和T * 的C-5间隔五或六个核苷酸单位。 计划进行研究以确定这些修改如何影响 与互补DNA和RNA杂交的特异性和强度 序列的 随后另外的修饰的寡核苷酸 与兔珠蛋白mRNA中的许多不同区域互补 将被合成并进行结合和切割实验。 最终，该方法可适用于控制 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）。 该方法的模块化性质使其 潜在地广泛适用于广泛的病毒。 如果 在此授权期间开发的模型化合物的功能是 预期，那么我们将能够设计寡核苷酸 靶向特定的HCMV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.