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。 使用真核细胞中的瞬时表达系统来观察 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 已发表(1987)，以及讨论技术的第二篇论文 关于使用反义寡核苷酸的研究已经发表(1988年)。 使用反义寡核苷酸的实验现在已经扩展到 以EBV基因表达为模型研究潜在的抗病毒作用。 反义分子似乎能早期抑制特定EBV的表达 佛波醇刺激B95-8细胞诱导的抗原EA(D) 免疫荧光法和酶联免疫吸附试验检测Ester-TPA 抗EA(D)抗体。正在对更多的反义序列进行检测 确定这种抑制的特异性。我们已经购买了一个 用于这些实验的合成寡核苷酸的仪器和J. 史密斯最近加入了实验室，这应该会对 提供材料，这些材料一直是该项目的一个限制因素。我们 已经开发出大规模合成反义的方案 寡核苷酸和高效液相纯化，并计划实施 在不久的将来。

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) as well as 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, EA (D), induced by stimulation of B95-8 cells with the phorbol ester TPA as assayed by immunofluorescence and ELISA using a monoclonal antibody to EA(D). Additional antisense sequences are being examined to determine the specificity of this inhibition. We have purchased an instrument to synthesize oligonucleotides for these experiments and J. Smith has recently joined the laboratory, which should help greatly to supply materials which have been a limitting factor in this project. We have developed protocols for largescale synthesis of antisense oligonucleotides and purification by HPLC, and implementation is planned in the near future.