RNA TARGETING WITH TERTIARY INTERACTIONS

描述：(改编自申请者摘要)这份R03申请在 对一项名为创新药物发现研究的计划公告的回应 在艾滋病机会性感染中。这个项目的最终目标是 发现允许使用小分子寡核苷酸靶向RNA的原理 抑制功能的基本化合物。确立这些原则可以 具有广泛的应用，包括设计治疗艾滋病毒的化合物和 机会性感染。有待探索的新范式有：(1)约束性 三级相互作用增强(Beti)，(2)自杀抑制，和(3) 寡核苷酸催化的剪接位点的水解性。第一组自我剪接 卡氏肺孢子虫核糖体RNA前体的内含子将作为 这些研究的模型体系。这个前体的正确拼接是 形成活性核糖体所必需的。初步工作表明， 核酸酶稳定，N3‘-P5’磷酰胺六核苷酸显示结合 通过与序列紧密结合的第三级相互作用增强 通常对齐5‘拼接点。这种六核苷酸抑制自剪接 通过自杀抑制机制。拟议工作的具体目标是： (1)确定不带电荷的甲基膦酸键的寡核苷酸 展示Beti和抑制自剪接。(2)判断Beti是否为 可在第一组内含子上的其他位置获得。(3)体外检测 寡核苷酸抑制剂被卡氏肺孢子虫细胞摄取。结果是 有望为设计许多RNA的抑制剂提供基本原则 功能。因为rrna前体的成熟是增殖所必需的。 卡氏肺孢子虫，在人类基因中没有已知的I组自剪接内含子， 这一结果也可能为卡氏肺孢子虫肺炎提供潜在的治疗方法。

DESCRIPTION: (Adapted from Applicant's Abstract) This RO3 application is in response to a Program Announcement entitled Innovative Drug Discovery Research in AIDS Opportunistic Infections. The ultimate goal of this project is to discover principles that allow targeting of RNA with small oligonucleotide based compounds that inhibit function. Establishment of such principles could have wide ranging applications, including design of compounds to treat HIV and opportunistic infections. New paradigms to be explored are (1) binding enhancement by tertiary interactions (BETI), (2) suicide inhibition, and (3) oligonucleotide-catalyzed hydrolysis of splice sites. The group I self-splicing intron from the ribosomal RNA precursor in Pneumocystis carinii will serve as the model system for these studies. Correct splicing of this precursor is required for formation of active ribosomes. Preliminary work has shown that a nuclease stable, N3'-P5' phosphoramidate hexanucleotide exhibits binding enhanced by tertiary interactions in binding tightly to the sequence that normally aligns the 5' splice site. This hexanucleotide inhibits self-splicing via a suicide inhibition mechanism. Specific aims for the proposed work are: (1) Determine if oligonucleotides with uncharged methylphosphonate linkages exhibit BETI and inhibition of self-splicing. (2) Determine if BETI is available at other sites on the group I intron. (3) Determine if in vitro oligonucleotide inhibitors are taken up by P. carinii cells. The results are expected to provide fundamental principles for designing inhibitors of many RNA functions. Because maturation of rRNA precursor is essential for proliferation of P. carinii, and no group I self-splicing introns are known in human genes, the results could also provide potential therapeutics for P. carinii pneumonia.