Modified single-stranded DNA oligonucleotides can direct nucleotide exchange in Saccharomyces cerevisiae. Point and frameshift mutations are corrected in a reaction catalyzed by cellular enzymes involved in various DNA repair processes. The present model centers on the annealing of the vector to one strand of the helix, followed by the correction of the designated base. The choice of which strand to target is a reaction parameter that can be controlled, so here we investigate the properties of strand bias in targeted gene repair. An in vivo system has been established in which a plasmid containing an actively transcribed, but mutated, hygromycinenhanced green fluorescent protein fusion gene is targeted for repair and upon conversion will confer hygromycin resistance on the cell. Overall transcriptional activity has a positive influence on the reaction, elevating the frequency. If the targeting vector is synthesized so that it directs nucleotide repair on the nontranscribed strand, the level of gene repair is higher than if the template strand is targeted. We provide data showing that the targeting vector can be displaced from the template strand by an active T7 phage RNA polymerase. The strand bias is not influenced by which strand serves as the leading or lagging strand during DNA synthesis. These results may provide an explanation for the enhancement of gene repair observed when the nontemplate strand is targeted.The use of oligonucleotides to direct single-base changes in DNA has become an important strategy for understanding gene function. While processes for disabling targeted genes and creating knockout strains have provided an enormous amount of information, it is now becoming evident that subtle changes in the genomes of many organisms constitute a centerpiece of genetic variability. Specifically, as information regarding the human genome accumulates, the importance of single-nucleotide polymorphisms is now clear. These simple alterations, often causing no visible phenotype variation, may hold the key to the spectrum of responses to many therapeutic agents. In recognition of the importance of single-nucleotide polymorphisms, several technologies have emerged that aim to create animal and plant models that will recapitulate the nucleotide alterations without disrupting the integrity of the whole gene. Many of these technologies employ synthetic oligonucleotides as vectors for directing the introduction of the specific changes.Our laboratory has developed a series of these oligonucleotide vectors that can create single-base changes in chromosomal and episomal targets. One of these vectors is the chimeric RNA/DNA oligonucleotide (chimera), which consists of cRNA and DNA residues folded into a double hairpin conformation. Chimeras have been shown to direct base replacement or base insertion in yeast (21, 27), mammalian cells in culture (1, 6, 15, 38), animal models (2, 14, 16, 26, 31), and plants (3,28,40). While the mechanism of targeted gene repair is not fully elucidated, a number of important steps have...

Targeted gene repair uses short DNA oligonucleotides to direct a nucleotide exchange reaction at a designated site in a mammalian chromosome. The widespread use of this technique has been hampered by the inability of workers to achieve robust levels of correction. Here, we present a mammalian cell system in which DLD-1 cells bearing integrated copies of a mutant eGFP gene are repaired by modified single-stranded DNA oligonucleotides. We demonstrate that two independent clonal isolates, which are transcribed at different levels, are corrected at different frequencies. We confirm the evidence of a strand bias observed previously in other systems, wherein an oligonucleotide designed to be complementary to the nontranscribed strand of the target directs a higher level of repair than one targeting the transcribed strand. Higher concentrations of cell oligonucleotides in the electroporation mixture lead to higher levels of correction. When the target cell population is synchronized into S phase then released before electroporation, the correction efficiency is increased within the entire population. This model system could be useful for pharmacogenomic applications of targeted gene repair including the creation of cell lines containing single-base alterations.

Locked nucleic acids (LNAs) are novel base modifications containing a methylene bridge uniting the 2'-oxygen and the 4'-carbon. In this study, LNA-modified single-stranded molecules directed the repair of single base mutations in a yeast chromosomal gene. Using a genetic assay involving a mutant hygromycin-resistance gene, correction of point and frameshift mutations was facilitated by vectors containing an LNA residue on each terminus. Increasing the number of LNA bases on each terminus reduced the correction frequency progressively. When the LNA vector is used in combination with a phosphorothioate-modified vector (74-mer), however, a high level of gene-repair activity occurs; hence, short LNA-based vectors can augment the activity of other types of targeting vectors. These data suggest that oligonucleotides containing locked nucleic acid residues can be used to direct single nucleotide exchange reactions in vivo.