Site-directed recombination via bifunctional PNA-DNA conjugates
Site-directed recombination via bifunctional PNA-DNA conjugates
复制标题
DOI：
10.1073/pnas.262556899
复制
发表时间：
2002-12-24
影响因子：
11.1
通讯作者：
Glazer, PM
中科院分区：
文献类型：
--
作者：
Rogers, FA;Vasquez, KM;Glazer, PM
位点特异性DNA结合分子为哺乳动物细胞的遗传操作提供了潜力。肽核酸（PNA）是一种DNA模拟物，其中嘌呤和嘧啶碱基连接到聚酰胺骨架上。PNA通过Watson-Crick碱基配对以高亲和力结合单链DNA，并且可以通过Hoogsteen结合DNA/PNA双链体形成三螺旋。能够进行链侵入和三链体形成的二聚体双PNA可以在靶DNA上形成钳结构。作为促进定点重组的策略，将双PNA偶联至与靶基因中的相邻区域同源的40-nt供体DNA片段。发现PNA-DNA缀合物在人无细胞提取物中介导与质粒底物的定点重组，导致报告基因中的突变以高于背景至少60倍的频率校正。当双PNA和供体DNA在没有共价键的情况下共混合时，也观察到诱导的位点特异性重组。此外，发现双PNA和双PNA-DNA缀合物在靶质粒中特异性地诱导DNA修复。发现PNA诱导的重组和PNA诱导的修复都依赖于核苷酸切除修复因子XPA（着色性干皮病互补组A蛋白）。这些结果表明，双链体DNA上PNA钳的形成产生了螺旋扭曲，强烈地引起DNA修复，从而使靶位点对重组敏感。使用PNA-DNA缀合物以定点方式促进重组的能力可以提供实现缺陷基因的靶向校正的有用策略。
Site-specific DNA binding molecules offer the potential for genetic manipulation of mammalian cells. Peptide nucleic acids (PNAs) are a DNA mimic in which the purine and pyrimidine bases are attached to a polyamide backbone. PNAs bind with high affinity to single-stranded DNA via Watson-Crick base pairing and can form triple helices via Hoogsteen binding to DNA/PNA duplexes. Dimeric bis-PNAs capable of both strand invasion and triplex formation can form clamp structures on target DNAs. As a strategy to promote site-directed recombination, a bis-PNA was coupled to a 40-nt donor DNA fragment homologous to an adjacent region in the target gene. The PNA-DNA conjugate was found to mediate site-directed recombination with a plasmid substrate in human cell-free extracts, resulting in correction of a mutation in a reporter gene at a frequency at least 60-fold above background, Induced site-specific recombination was also seen when the bis-PNA and the donor DNA were co-mixed without covalent linkage. In addition, the bis-PNA and the bis-PNA-DNA conjugate were found to induce DNA repair specifically in the target plasmid. Both the PNA-induced recombination and the PNA-induced repair were found to be dependent on the nucleotide excision repair factor, XPA (xeroderma pigmentosum complementation group A protein). These results suggest that the formation of a PNA clamp on duplex DNA creates a helical distortion that strongly provokes DNA repair and thereby sensitizes the target site to recombination. The ability to promote recombination in a site-directed manner using PNA-DNA conjugates may provide a useful strategy to achieve targeted correction of defective genes.