INHIBITION OF GENE EXPRESSION BY NORMAL & MODIFIED ANTISENSE OLIGONUCLEOTIDES

这个项目最初是为了确定是否

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.