Havre P A, Gunther E J, Gasparro F P, Glazer P M
Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT 06510.
Proc Natl Acad Sci U S A. 1993 Aug 15;90(16):7879-83. doi: 10.1073/pnas.90.16.7879.
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.
寡核苷酸可以以序列特异性方式在双链DNA的同型嘌呤/同型嘧啶区段的大沟中作为第三条DNA链结合。在此，我们使用一个在其5'端连接了补骨脂素衍生物的10碱基三链体形成寡核苷酸，以在完整的双链λ噬菌体基因组中实现位点特异性的靶向诱变。位点特异性三链体的形成将补骨脂素递送至λDNA中的靶向位点，补骨脂素的光激活产生加合物，从而在该位点产生突变。靶向基因中的突变频率比非靶向基因中的至少高100倍，并且靶向基因中突变的序列分析表明，96%的突变位于靶向区域，并且发现56%的突变恰好是在靶向碱基对处发生相同的T.A到A.T颠换。通过使用修饰的寡核苷酸将突变可重复且可预测地靶向完整双链DNA中的位点的能力，可能作为一种基因治疗技术、一种抗病毒治疗方法以及一种基因工程工具被证明是有用的。
