Control of the expression of an early gene of SV-40 with natural and modified oligonucleotides

On the basis of the literature we thought that an antisense sequence carrying L-2'-deoxynucleosides in the terminal or next to the terminal position should be more resistent toward exonucleases, thus more effective than the natural analog, if the duplex formed with the target m-RNA have comparable stabilities. This hypothesis was tested employing the following oligonucleotides: AACTTTATCCATC (NA), (L_dA)ACTTTATCCA(L-dT)C (MA) and (L-dA)TGGATAAAGT(L-dT)T MS; which have as a target the first four tranlatedcodons of the m-RNA derived from the T grne of SV-40. The oligomers were tested, in different concentrations, as inhibitors of sv-40 replication in VERO cells infected with a m.o.i. of 20 PFU/cell. Forty hrs after infections cells were lysed and the viral DNA extracted and analysed by agarose gel electrophoresis. DNA synthesis inhibition was quantified by densitometric scanning of ethidium bromide stained gels or by southern-blot hybridization, using a full-lenght genomic SV-40 probe. A concentration of 200 mg/ml of MA completely inhibited the SV-40 replication as measured by viral DNA production at 40 hrs post-infection while at the same dose MS and NA produced a markedly lower inhibition. In the last two cases reduction of viral replication is most likely connected with cytotoxicity (equal to 45%, 44% and 30% for MA; MS and NA respectively at 200 mg/ml). The modified sequences could be labelled with 32P by T4 polynucleotide kinase to the same extent of natural compounds.