INHIBITION OF GENE EXPRESSION BY NORMAL & MODIFIED ANTISENSE OLIGONUCLEOTIDES

该项目最初是为了确定是否 适当反义序列的修饰寡核苷酸可以抑制 生物反应，并研究其作为抗病毒药物的潜力。一个 利用真核细胞中的瞬时表达系统观察 正常寡核苷酸和修饰的寡核苷酸均能抑制酶的活性。 介绍了氯霉素转移酶CAT(CAT基因) 通过DNA转染法进入细胞)。经测定，微摩尔 修饰的寡核苷酸浓度可抑制CAT活性和 化学修饰的链长、浓度和性质 寡核苷酸的多少可以影响抑制的程度。这部作品 已经发表了(1987)，最近又发表了第二篇讨论技术的论文 关于使用反义寡核苷酸的研究已经发表(1988年)。实验 使用反义寡核苷酸现已扩展到研究潜在的 以EBV基因表达作为抗病毒作用的模型。反义分子 似乎抑制了特定的EBV早期抗原F-A(D)的表达， 佛波酯TPA对B95-8细胞的刺激诱导 用F-A(D)的单抗进行免疫荧光和酶联免疫吸附试验。 正在检测更多的反义序列以确定 这种抑制的特异性。我们最近购买了一台仪器来 为这些实验合成寡核苷酸，这应该会有很大帮助 以提供材料，这些材料一直是该项目的限制因素。 在过去的一年里，我们开发了用于大规模合成的协议 反义寡核苷酸的制备及高效液相色谱纯化。

This project was initially undertaken in order to determine whether modified oligonucleotides of appropriate antisense sequence can inhibit biological reactions, and to examine their potential as antiviral agents. A transient expression system in eukaryotic cells was used to see whether normal and modified oligonucleotides could inhibit the activity of the enzyme chloramphenicol transferase CAT (the gene for CAT was introduced into the cells by DNA transfection). It was determined that micromolar concentrations of modified oligonucleotides could inhibit CAT activity and that chain length, concentration, and nature of the chemical modification of the oligonucleotide could influence the degree of inhibition. This work has been published (1987) and recently a second paper discussing techniques for using antisense oligonucleotides has been published (1988). Experiments using antisense oligonucleotides have now been extended to study potential antiviral effects using EBV gene expression as a model. Antisense molecules appear to inhibit expression of a specific EBV early antigen, F-A(D), induced by stimulation of B95-8 cells with the phorbol ester TPA as assayed by immunofluorescence and ELISA using a monoclonal antibody to F-A(D). Additional antisense sequences are being examined to determine the specificity of this inhibition. We have recently purchased an instrument to synthesize oligonucleotides for these experiments which should help greatly to supply materials which have been a limiting factor in this project. During the past year we have developed protocols for large-scale synthesis of antisense oligonucleotide and purification by HPLC.