Gene correction by 5'-tailed duplexes with short editor oligodeoxyribonucleotides.

Various diseases, including cancer, are caused by genetic mutations. A 5'-tailed duplex (TD) DNA, consisting of a long single-stranded (ss) editor DNA and a short (∼35-base) ss assistant oligodeoxyribonucleotide, can introduce a base-substitution in living cells and thus correct mutated genes. Previously, several hundred-base DNAs were employed as the editor DNAs. In this study, 5'-TDs were prepared from various editor DNAs with different lengths and examined for their gene correction abilities, using plasmid DNA bearing a mutated copepod green fluorescent protein (copGFP) gene, in human cells. High-throughput analysis was performed by the reactivated fluorescence of the wild-type protein encoded by the corrected gene as the indicator. The analysis revealed that 5'-TDs with ∼100-base ss editor DNAs enabled gene editing at least as efficiently as those with longer editor DNAs. Moreover, the antisense strand was more effective as the editor than the sense strand, in contrast to the 5'-TDs with longer editor strands. These results indicated that the 5'-TD fragments with shorter editor strands than those used in previous studies are useful nucleic acids for gene correction.

Effects of mismatches distant from the target position on gene correction with a 5'-tailed duplex.

The introduction of a 5'-tailed duplex (5'-TD) fragment into cells corrects a base-substitution mutation in a target DNA. We previously reported that the gene correction efficiency was improved when a frameshift type of second mismatch was present ∼330 bases distant from the target position, between the target DNA and the 5'-TD fragment. In this study, the effects of the second mismatches on the gene correction were further examined. Base-base mismatches 332 bases distant from the target position slightly enhanced gene correction, but less efficiently than the previously studied frameshift mismatches. The gene correction efficiency was also increased when the distance between the target position and the second frameshift mismatch was changed to ∼270 bases. These results suggested that the introduction of an appropriate second frameshift mismatch into the 5'-TD fragment improves the gene correction efficiency.