Gamper H B, Cole-Strauss A, Metz R, Parekh H, Kumar R, Kmiec E B
Department of Biological Sciences, University of Delaware, Newark, Delaware 19716, USA.
Biochemistry. 2000 May 16;39(19):5808-16. doi: 10.1021/bi9921891.
Self-complementary chimeric oligonucleotides that consist of DNA and 2'-O-methyl RNA nucleotides arranged in a double-hairpin configuration can elicit a point mutation when targeted to a gene sequence. We have used a series of structurally diverse chimeric oligonucleotides to correct a mutant neomycin phosphotransferase gene in a human cell-free extract. Analysis of structure-activity relationships demonstrates that the DNA strand of the chimeric oligonucleotide acts as a template for high-fidelity gene correction when one of its bases is mismatched to the targeted gene. By contrast, the chimeric strand of the oligonucleotide does not function as a template for gene repair. Instead, it appears to augment the frequency of gene correction by facilitating complex formation with the target. In the presence of RecA protein, each strand of a chimeric oligonucleotide can hybridize with double-stranded DNA to form a complement-stabilized D-loop. This reaction, which may take place by reciprocal four-strand exchange, is not observed with oligonucleotides that lack 2'-O-methyl RNA segments. Preliminary sequencing data suggest that complement-stabilized D-loops may be weakly mutagenic. If so, a low level of random mutagenesis in the vicinity of the chimera binding site may accompany gene repair.
由DNA和以双发夹结构排列的2'-O-甲基核糖核苷酸组成的自互补嵌合寡核苷酸靶向基因序列时可引发点突变。我们使用了一系列结构多样的嵌合寡核苷酸在人无细胞提取物中校正突变的新霉素磷酸转移酶基因。结构-活性关系分析表明，当嵌合寡核苷酸的一条链的一个碱基与靶向基因错配时，其DNA链可作为高保真基因校正的模板。相比之下，寡核苷酸的嵌合链不能作为基因修复的模板。相反，它似乎通过促进与靶标的复合物形成来提高基因校正的频率。在RecA蛋白存在的情况下，嵌合寡核苷酸的每条链可与双链DNA杂交形成互补稳定的D环。这种反应可能通过相互的四链交换发生，缺乏2'-O-甲基RNA片段的寡核苷酸不会出现这种反应。初步测序数据表明，互补稳定的D环可能具有弱诱变作用。如果是这样，在嵌合体结合位点附近可能会伴随基因修复出现低水平的随机诱变。
