Effects of Target Sequence and Sense versus Anti-sense Strands on Gene Correction with Single-stranded DNA Fragments

Abstract

SummaryThe correction of an inactivated hygromycin-resistance and enhanced green fluorescent protein (Hyg-EGFP) fusion gene by a several hundred-base single-stranded (ss) DNA fragment has been reported. In this study, the effectiveness of this type of gene correction was examined for various positions in the rpsL gene. Sense and antisense ss DNA fragments were prepared, and the gene correction efficiencies were determined by co-introduction of the target plasmid containing the gene with the ss DNA fragments. The gene correction efficiency varied (0.8-9.3%), depending on target positions and sense/antisense strands. Sense ss DNA fragments corrected the target gene with equal or higher efficiencies as compared to their antisense counterparts. The target positions corrected with high efficiency by the sense fragments also tended to be corrected efficiently by the antisense fragments. These results suggest that the sense ss DNA fragments are useful for the correction of mutated genes. The variation in the correction efficiency may depend on the sequence of the target position in double-stranded DNA.Key Words: gene correction; single-stranded DNA fragment; nucleic acid therapeutics; genetic engineering; rpsL gene. 3Gene correction (nucleotide sequence conversion), by which a mutated gene is converted to one with the normal (or desired) sequence, is an attractive strategy for gene therapy (1-10). Disruption of a gene involved in maintenance of a disease could be conducted with this technology. The corrected genes can be expressed under the control of their authentic regulatory elements. Moreover, gain-of-function or dominant mutations, such as activated oncogenes, could be suitable subjects for the gene correction strategy. Various kinds of devices, such as ss oligonucleotides, triplex-forming oligonucleotides, and heat-denatured, 400-800 bp ds PCR fragments, have been examined as nucleic acid tools for gene correction (1-10).Previously, it was reported that a several hundred-base ss DNA fragment containing the sense sequence, prepared by restriction enzyme digestions of ss phagemid DNA, corrected an inactivated episomal Hyg-EGFP fusion gene with higher gene correction efficiency, in comparison with the conventional PCR fragment (11). In contrast, the correction with the ss DNA fragment containing the antisense sequence was less efficient than that with the sense ss DNA fragment.These results raised the questions of whether ss DNA fragments could be used to correct different target sequences and whether sense fragments are more efficient than the corresponding antisense fragments for other target sequences. In this study, we chose the rpsL (bacterial streptomycin-resistance) gene as the target gene. The gene encodes the S12 protein, one of the small subunit proteins of the E. coli ribosome. Specific mutations at various positions in the rpsL gene confer streptomycin-resistant phenotype (strA) to bacteria. The E. coli strA strain harboring the plasmid with the wild-type rpsL gene exhibits the streptomycin-sensitive phe...

Cleavage of Target DNA Promotes Sequence Conversion with a Tailed Duplex

“…[10][11][12][13] In this study, we used the lacZα gene from a cloning vector (M13mp18), since sequence conversion from the mutated (TAG) sequence to the "wild-type" (TCG) could be easily judged by the blue-white selection and the PstI and XbaI sites are located near the target position (Fig. 2B).…”

Insertion and Deletion Mismatches Distant from the Target Position Improve Gene Correction with a Tailed Duplex

“…The bacteria were seeded onto LB agar plates containing 200 µg/ml of streptomycin and 50 µg/ml of kanamycin (selection plates) and LB agar plates containing 50 µg/ml of kanamycin (titer plates), as described previously. [18] Gene correction efficiencies were calculated by dividing the number of streptomycin-resistant colonies on the selection plates by the number of colonies on the titer plates.…”

“…[10,11,18] In this study, we compared the gene correction (sequence alteration) efficiencies when indel mismatches were present ~330 bases distant from the target position. The target base was T at position 239 (the 2nd position of codon 80, ATC) in the pSSW plasmid DNAs, and the corresponding base was G in the TD fragment (AGC) (Fig.…”

Correction of Frameshift Mutations with Tailed Duplex DNAs

“…[8][9][10] However, the ss DNA fragment corrects frameshift (singlebase deletion and single-base insertion) mutations with low efficiencies.…”

“…[2][3][4][5][6][7] We previously reported that a single-stranded (ss) DNA fragment containing the sense sequence, and tailed duplex (TD) DNA fragments prepared by annealing an oligonucleotide to the ss DNA fragment, have the ability to correct single-base substitution mutations. [8][9][10] However, the ss DNA fragment corrects frameshift (singlebase deletion and single-base insertion) mutations with low efficiencies. 11) Since the correction efficiencies of single-base substitution mutations by the TD fragments are higher than those by the ss DNA fragment, 10) it is important to examine the ability of the TD fragments to correct frameshift mutations.…”