Abstract
BackgroundTransfection of cells with gene-specific, single-stranded oligonucleotides can induce the targeted exchange of one or two nucleotides in the targeted gene. To characterize the features of the DNA-repair mechanisms involved, we examined the maximal distance for the simultaneous exchange of two nucleotides by a single-stranded oligonucleotide. The chosen experimental system was the correction of a hprt-point mutation in a hamster cell line, the generation of an additional nucleotide exchange at a variable distance from the first exchange position and the investigation of the rate of simultaneous nucleotide exchanges.ResultsThe smaller the distance between the two exchange positions, the higher was the probability of a simultaneous exchange. The detected simultaneous nucleotide exchanges were found to cluster in a region of about fourteen nucleotides upstream and downstream from the first exchange position.ConclusionWe suggest that the mechanism involved in the repair of the targeted DNA strand utilizes only a short sequence of the single-stranded oligonucleotide, which may be physically incorporated into the DNA or be used as a matrix for a repair process.
Highlights
Transfection of cells with gene-specific, single-stranded oligonucleotides can induce the targeted exchange of one or two nucleotides in the targeted gene
We suggest that if DNA sequence analysis of transfected cells shows the exchange of both targeted nucleotides, at least the region of the oligonucleotide situated between the two mismatches has been used for TNE
Investigation of the minimal distance of the second mismatch nucleotide from the 3'-end of the oligonucleotides The oligonucleotides used were delivered into the cell by magnet-assisted transfection
Summary
Transfection of cells with gene-specific, single-stranded oligonucleotides can induce the targeted exchange of one or two nucleotides in the targeted gene. To characterize the features of the DNA-repair mechanisms involved, we examined the maximal distance for the simultaneous exchange of two nucleotides by a single-stranded oligonucleotide. Transfection of cells with single-stranded oligonucleotides showing a mismatch to a target gene sequence can result in an exchange of the single nucleotide in the genomic DNA [1,2,3,4,5,6,7,8]. Proteins involved in mismatch repair (MMR) seem to be crucial for this nucleotide exchange in yeast but not in mammalian cells [19]. Another possible repair mechanism involved in this step is nucleotide exchange repair [20]. The alteration of the sequence (page number not for citation purposes)
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