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.
