MOLECULAR STUDIES OF GENE TARGETED OLIGONUCLEOTIDES

基因靶向寡核苷酸的分子研究

• 批准号: 2740016
• 负责人: Cy A STEIN
• 金额: $ 34.73万
• 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/2740016
• 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.

反义技术在理论上为人类提供了一种极好的方法， 验证基因功能。然而，在使用中存在显著的问题， 全硫代磷酸骨架寡核苷酸的。这些问题 由电荷、硫含量和非序列特异性引起。我们 假设降低电荷密度可以改善这些情况。 因此，在特定目标1中，我们将合成含有以下成分的寡聚物 部分带电的甲基膦酸酯（MP）/磷酸二酯交替 (PO)和硫代磷酸酯（PS）主链（alt-OMP）。反义alt-OMPs 含有2 ′-O-甲基核糖核苷（alt-mr-OMP）， 与单链RNA靶点相互作用。将制定方法， 制备具有限定构型的MP键的alt-mr-OMP。嵌合 寡聚体（chi-OMP），其在寡聚体的3'和5'-末端具有MP键， 还将检查低聚物和内部PO或PS链。为了 为了定量非序列特异性，我们将确定一个等级顺序， 寡核苷酸的非序列特异性通过它们与Mac-1结合的能力。 将在两种寡核苷酸之间进行比较，反义c-myb， 裸递送的反义PKC-α（Isis 3521）， 用一种新型的载体阳离子卟啉我们将在具体 目的2，在mRNA水平区分“真”反义与毒性： 1)使用有规立构的PS寡聚物， 将3'末端PS裂解为核苷酸单硫代磷酸：MP 还将检查定义的立体化学的键; 2）我们将 开发了一种功能性方法来确定alt-mr-OMPs与 它们在活细胞中的mRNA靶点。程序将使用主干- 优化的、生物素化的、紫杉醇衍生的alt-mr-OMP， 设计成与mRNA形成共价加合物。由此产生的限制 连接到alt-mr-OMP的片段将被捕获在 链霉亲和素珠。捕获的片段将通过PCR扩增， 一组特异性引物。3)确定c-myb oligo是否具有更高的 顺序结构，并导致改变的速度内体 流出我们将这与特异性Aim 1寡核苷酸的能力相关联， 从核内体中逃脱这些实验将由 共聚焦显微镜技术在第三个目标中，我们将 通过浓缩来增强alt-OMP和chi-OMP向细胞的递送 它们与一种新的传递剂，四（N-甲基吡啶基）卟啉。