Synthesis and evaluation of a triplex-forming oligonucleotide-pyrrolobenzodiazepine conjugate.
Synthesis and evaluation of a triplex-forming oligonucleotide-pyrrolobenzodiazepine conjugate.
复制标题
三链体形成寡核苷酸-吡咯并苯二氮卓缀合物的合成和评估。
DOI：
10.1021/bc0498673
复制
发表时间：
2004
期刊：
影响因子：
--
通讯作者：
Ebbinghaus,ScotW
中科院分区：
文献类型：
--
作者：
Zhilina,ZhannaV;Ziemba,AmyJ;Trent,JohnO;Reed,MichaelW;Gorn,Vladimir;Zhou,Qun;Duan,Wenhu;Hurley,Laurence;Ebbinghaus,ScotW
在大多数情况下，被设计为抗基因分子的未修饰的寡核苷酸不能以足够的稳定性与DNA结合以阻止基因表达。为了稳定与HER-2/neu启动子中的多嘌呤段的结合，将三链体形成寡核苷酸（TFO）缀合至吡咯并[1，4]苯并二氮杂卓（PBD）、去甲基托马霉素，并评价位点特异性DNA结合。PBD部分的活化酯通过酰化反应与TFO的5'端的50个原子的脂族接头上的游离伯胺缀合。设计该长脂肪族接头以提供从TFO的大沟结合位点到PBD的小沟结合位点的桥。与未缀合的TFO相比，通过所得的TFO−PBD缀合物形成三链体的速度更慢，亲和力低近30倍。PBD结合的三链体的目标被证明由限制性内切酶消化的保护，和共价结合的鸟嘌呤的环外氨基推断取代特定的鸟嘌呤与肌苷。虽然TFO的结合效率较低，但本报告表明，原则上，TFO可用于指导PBD与特定位置的结合。进一步优化TFO-PBD偶联物设计，可能涉及优化接头，可能在TFO的两端放置PBD，将需要使基因修饰稳健。
In most cases, unmodified oligonucleotides designed as antigene molecules are incapable of binding to DNA with sufficient stability to prevent gene expression. To stabilize binding to a polypurine tract in the HER-2/neu promoter, a triplex forming oligonucleotide (TFO) was conjugated to a pyrrolo[1,4]benzodiazepine (PBD), desmethyltomaymycin, and site-specific DNA binding was evaluated. An activated ester of the PBD moiety was conjugated by an acylation reaction to a free primary amine on a 50-atom aliphatic linker at the 5‘ end of the TFO. This long aliphatic linker was designed to provide a bridge from the major groove binding site of the TFO to the minor groove binding site of the PBD. Triplex formation by the resulting TFO−PBD conjugate occurred more slowly and with a nearly 30-fold lower affinity compared to an unconjugated TFO. PBD binding to the triplex target was demonstrated by protection from restriction enzyme digestion, and covalent binding to the exocyclic amino group of guanine was inferred by substituting specific guanines with inosines. Although the binding of the TFO was less efficient, this report demonstrates that in principle, TFOs can be used to direct the binding of a PBD to specific location. Further optimization of TFO−PBD conjugate design, likely involving optimization of the linker and perhaps placing a PBD at both ends of the TFO, will be needed to make gene modification robust.