Simultaneous targeted exchange of two nucleotides by single-stranded oligonucleotides clusters within a region of about fourteen nucleotides

Abstract

Background: 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. 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.

Results: The 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.

Conclusion: We 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.

Figure 1

Structure of the oligonucleotides and relative position of the two mismatches. (A) Sequence and structure of the oligonucleotides modified with a TA-clamp generating a first nucleotide exchange at position 153 (light blue) ore 151 (dark blue) respectively. The possible positions for the second (silent) exchange are given in red. (B) Structure of the oligonucleotides showing the first mismatch at position 153 and the second mismatch at position 147 in various distance from the unmodified 3'-end. (C) Structure of the oligonucleotides with different length, modified with PTO and showing the first mismatch at position 151 and the second mismatch at position 159.

Figure 2

Sequence analyses of hprt. Sequence analysis of the hprt gene isolated from V79-151 cell clones after transfection with oligonucleotide C59-153-147 (A) and H3-151-147 (B). Arrows point to the position of the successfully exchanged nucleotides. The upper base code shows the sequence of the analyzed clones, the lower base code the original sequence of the transfected V79-151 cells.

Figure 3

Correction rate of the first mismatch position. Correction rate of the first mismatch position after transfection with TA-clamp modified oligonucleotides carrying one mismatch (striped column) or two mismatches (black columns). The first exchange nucleotide was at hprt position 151 and the second mismatch at various distances. The X-axis indicates the distance of the second mismatch from the first exchange position in upstream and downstream direction. The Y-axis shows the mean number of clones obtained per 10⁶ transfected cells. Mean and standard deviations were calculated based on the total number of clones after 3 – 6 transfection experiments per oligonucleotide (Additional file 1).

Figure 4

Exchange rate of the second nucleotide. Percentage of clones with both exchanges after transfection with TA-clamp modified oligonucleotides carrying their first exchange nucleotide at hprt position 151. The X-axis indicates the distance of the second mismatch from the first exchange position in upstream and downstream direction.

Figure 5

Fitted exchange rate of the second nucleotide after transfection with differently modified oligonucleotides. Percentage of clones with both exchanges after different transfection experiments with oligonucleotides carrying two mismatches to hprt. The X-axis indicates the distance of the second mismatch (independent of its position in upstream or downstream direction) from the first exchange position. ● first mismatch at position 151, modified by TA-clamps. ◀ : first mismatch at position 153, modified by TA-clamps. ■ : first mismatch at position 151, modified by PTO. The black line shows the linear fit of the data points from the experiments with the three different kinds of oligonucleotides.