Targeted mutagenesis of DNA using triple helix-forming oligonucleotides linked to psoralen.

Oligonucleotides can bind as third strands of DNA in a sequence-specific manner in the major groove in homopurine/homopyrimidine stretches in duplex DNA. Here we use a 10-base triplex-forming oligonucleotide linked to a psoralen derivative at its 5' end to achieve site-specific, targeted mutagenesis in an intact, double-stranded lambda phage genome. Site-specific triplex formation delivers the psoralen to the targeted site in the lambda DNA, and photoactivation of the psoralen produces adducts and thereby mutations at that site. Mutations in the targeted gene were at least 100-fold more frequent than those in a nontargeted gene, and sequence analysis of mutations in the targeted gene showed that 96% were in the targeted region and 56% were found to be the same T.A to A.T transversion precisely at the targeted base pair. The ability to reproducibly and predictably target mutations to sites in intact duplex DNA by using modified oligonucleotides may prove useful as a technique for gene therapy, as an approach to antiviral therapeutics, and as a tool for genetic engineering. IMAGES:

寡核苷酸（Oligonucleotide）可通过序列特异性结合方式，结合于双链DNA的同嘌呤/同嘧啶序列区域的大沟中，充当DNA的第三条链。本研究使用一条10碱基的三链形成寡核苷酸，其5'端连接有补骨脂素（psoralen）衍生物，以此在完整的双链λ噬菌体（lambda phage）基因组中实现位点特异性靶向诱变。位点特异性的三链形成会将补骨脂素递送至λDNA的靶位点，补骨脂素经光活化后会形成加合物，进而在该位点诱发突变。靶基因中的突变频率至少比非靶基因高100倍；对靶基因突变的序列分析显示，96%的突变位于靶区域内，且56%的突变均为精准发生于靶碱基对位置的T·A到A·T颠换。通过使用修饰型寡核苷酸，将突变可重复且可预测地靶向至完整双链DNA的特定位点的能力，有望作为基因治疗技术、抗病毒治疗手段以及基因工程工具得到应用。图像：