TARGETED MUTAGENESIS OF SIMIAN VIRUS-40 DNA-MEDIATED BY A TRIPLE HELIX-FORMING OLIGONUCLEOTIDE

Triple-helical DNA can be formed by oligonucleotides that bind as thir d strands of DNA in a sequence-specific manner in the major groove in homopurine/homopyrimidine stretches in duplex DNA. Such triple helix-f orming oligonucleotides have been used to inhibit gene expression by b locking transcription factor access to promoter sites in transient exp ression assays. In an alternative approach to genetic manipulation usi ng triplex DNA, we show that triplex-forming oligonucleotides can be u sed to produce site-specific, targeted mutations in a viral genome in order to achieve a permanent, heritable effect on gene function and ex pression. We use a triplex-forming oligonucleotide linked to a psorale n derivative at its 5' end to achieve targeted mutagenesis in a simian virus 40 (SV40) vector genome. Site-specific triplex formation delive rs the psoralen to the targeted site in the SV40 DNA. Photoactivation of the psoralen yields adducts and thereby mutations at that site. Mut ations were produced in the target gene in over 6% of the viral genome s. DNA sequence analysis of the mutations in the target gene showed th at all were in the targeted region, and 55% were found to be the same T:A-to-A:T transversion precisely at the targeted base pair. In contro l experiments, no mutagenesis above the background frequency in the as say was produced by a non-triplex-forming, psoralen-linked oligonucleo tide unless a vast excess of this oligonucleotide was used, demonstrat ing the specificity of the targeted mutagenesis. This frequency of tar geted mutagenesis of SV40 in monkey cells represents a 30-fold increas e relative to similar experiments using lambda phage in bacteria, sugg esting that fixation of the triplex-directed lesion into a mutation oc curs more efficiently in mammalian cells. If the ability to reproducib ly and predictably target mutations to sites in viral DNA in vitro by using modified oligonucleotides can be extended to DNA in vivo, this a pproach may prove useful as a technique for gene therapy, as a strateg y for antiviral therapeutics, and as a tool for genetic engineering.