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 third strands of DNA in a sequence-specific manner in the major groove in homopurine/homopyrimidine stretches in duplex DNA. Such triple helix-forming oligonucleotides have been used to inhibit gene expression by blocking transcription factor access to promoter sites in transient expression assays. In an alternative approach to genetic manipulation using triplex DNA, we show that triplex-forming oligonucleotides can be used to produce site-specific, targeted mutations in a viral genome in order to achieve a permanent, heritable effect on gene function and expression. We use a triplex-forming oligonucleotide linked to a psoralen derivative at its 5' end to achieve targeted mutagenesis in a simian virus 40 (SV40) vector genome. Site-specific triplex formation delivers the psoralen to the targeted site in the SV40 DNA. Photoactivation of the psoralen yields adducts and thereby mutations at that site. Mutations were produced in the target gene in over 6% of the viral genomes. DNA sequence analysis of the mutations in the target gene showed that 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 control experiments, no mutagenesis above the background frequency in the assay was produced by a non-triplex-forming, psoralen-linked oligonucleotide unless a vast excess of this oligonucleotide was used, demonstrating the specificity of the targeted mutagenesis. This frequency of targeted mutagenesis of SV40 in monkey cells represents a 30-fold increase relative to similar experiments using lambda phage in bacteria, suggesting that fixation of the triplex-directed lesion into a mutation occurs more efficiently in mammalian cells. If the ability to reproducibly and predictably target mutations to sites in viral DNA in vitro by using modified oligonucleotides can be extended to DNA in vivo, this approach may prove useful as a technique for gene therapy, as a strategy for antiviral therapeutics, and as a tool for genetic engineering.
Havre
P A
PA
Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, Connecticut 06510.
Glazer
P M
PM
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D004247:DNA; D005363:Ficusin; D005814:Genes, Viral / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D017422:Sequence Analysis, DNA; D013539:Simian virus 40 / Q000235:genetics*
D009838:Oligodeoxyribonucleotides; C051802:triplex DNA; D004247:DNA; D005363:Ficusin