MOLECULAR STUDIES OF GENE TARGETED OLIGONUCLEOTIDES

基因靶向寡核苷酸的分子研究

• 批准号: 6351281
• 负责人: Cy A STEIN
• 金额: $ 34.69万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-02-01 至 2003-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6351281
• 关键词: antisense nucleic acid; binding proteins; gel electrophoresis; gene targeting; glioma; messenger RNA; neoplastic cell; oligonucleotides; phosphonate; phosphoric ester; polymerase chain reaction; protein kinase C; protein structure; protooncogene; ribonucleosides; stereochemistry; thiophosphate

The antisense technology in theory provides a superb method for validating gene function. However, significant problems exist in the use of the all-phosphorothioate backbone oligonucleotides. These problems result from charge, sulfur content, and non-sequence specificity. We hypothesize that reducing charge density can ameliorate these. Therefore, in Specific Aim 1 we will synthesize oligos which contain partially charged, alternating methyklphosphonate (MP)/phosphodiester (PO) and phosphorothioate (PS) backbones (alt-OMPs). Antisense alt-OMPs contain 2'-O-methylribonucleosides (alt-mr-OMPs) and are designed to interact with single-stranded RNA targets. Methods will be developed to prepare alt-mr-OMPs with MP linkages of defined configuration. Chimeric oligomers (chi-OMPs) having MP linkages at the 3' and 5'-ends of the oligomer and internal PO or PS linages will also be examined. In order to quantitate non sequence specificity, we will determine a rank order of oligo non-sequence specificity by their ability to bind to Mac-1. Comparisons will be made between two oligos, antisense c-myb, which is delivered naked, and antisense PKC-alpha (Isis 3521) which is delivered with a novel vehicle, a cationic porphyrin. We will then, in Specific Aim 2, discriminate "true" antisense at the mRNA level from toxicity by: 1) using stereoregular PS oligos because of differential nuclease cleavage of the 3' terminal PS to a nucleotide monothiophosphate: MP linkages of defined stereochemistry will also be examined; 2) We will develop a functional method to determine of alt-mr-OMPs interact with their mRNA targets in living cells. The procedure will use backbone- optimized, biotinylated, psoralen-derivatized alt-mr-OMPs which are designed to form a covalent adduct with mRNA. The resulting restriction fragment which is attached to the alt-mr-OMP will be captured on streptavidin beads. The captured fragment will be amplified by PCR using a set of specific primers. 3) Determining if the c-myb oligo has higher order structure, and leads to alterations in the rate of endosomal efflux. We will correlate this with the ability of Specific Aim 1 oligos to escape from endosomes. These experiments will be performed by a confocal microscopic technique. Finally, in Specific Aim 3, we will amplify the delivery of alt-OMPs and chi-OMPs to cells by condensing them with a novel delivery agent, tetra (N-methylpyridyl) porphine.

理论上的反义技术提供了一种极好的方法 验证基因功能。然而，在使用中存在着重大问题。 全硫代主链寡核苷酸。这些问题 由电荷、硫含量和非序列特异性所致。我们 假设降低电荷密度可以改善这些。 因此，在特定的目标1中，我们将合成包含 部分带电的交替甲基磷酸盐(MP)/磷酸二酯 (PO)和(PS)主干(ALT-OMPS)。反义ALT-OMPS 含有2‘-O-甲基核糖核苷(ALT-MR-OMPS)，旨在 与单链RNA靶标相互作用。方法将被开发为 准备具有定义配置的MP链接的ALT-MR-OMPS。嵌合体 在3‘和5’端具有MP键的齐聚物(CHI-OMPS) 还将检查齐聚物和内部PO或PS衬里。按顺序 为了量化非序列特异性，我们将确定一个等级顺序 通过它们与Mac-1结合的能力而具有寡聚非序列特异性。 将对两种寡核苷酸进行比较，反义c-myb，它是 裸体和反义PKC-α(ISIS 3521) 用一种新的载体，阳离子卟啉。然后，我们将具体地 目的2，通过以下方法在信使核糖核酸水平上区分“真正的”反义和毒性： 1)由于核酸酶的差异，使用立体规则的PS寡聚糖 3‘端PS裂解成核苷酸一硫代磷酸：MP 还将审查已定义的立体化学的联系；2)我们将 建立测定ALT-MR-OMPS相互作用的功能方法 它们的信使核糖核酸以活细胞为靶标。该程序将使用主干- 优化的、生物素化的、补骨脂素衍生化的ALT-MR-OMPS 被设计成与信使核糖核酸形成共价加合物。由此产生的限制 附加到ALT-MR-OMP的片段将在 链霉亲和素珠子。捕获的片段将通过使用 一套特定的引子。3)确定c-myb寡核苷酸是否具有更高的 有序结构，并导致内体速率的变化 外流。我们会将其与特定目标1寡核苷酸的能力相关联 从内体中逃脱。这些实验将由一名 共聚焦显微镜技术。最后，在具体目标3中，我们将 通过浓缩扩增ALT-OMPS和CHI-OMPS到细胞的递送 它们与一种新型的递送剂，四(N-甲基吡啶)卟啉。