Design of Gene Therapeutic Agent Possessing Cooperative DNA Alkylating Ability

序列特异性DNA烷基化由于其在分子生物学和人类医学方面的巨大潜力，一直是当前非常感兴趣的话题。本文建立了合成多霉素A （Du）与n -甲基咪唑(lm)- n -甲基吡咯（Py）发夹聚酰胺新型偶联物及其DNA烷基化的一般方法。根据Dervan的环配对规则，设计了共轭物PyPyPygammalmPyPyDu （8a）和JmPyPygammalmPyPyDu （8b）分别对目标序列（A/T）G(A/T)_2N（A/G）和（A/T）G(A/T)CN（A/G）进行烷基化。高分辨率变性凝胶电泳结果表明，8a完全烷基化了-400 bp DNA片段中5‘-TGTAAAA-3’序列的A。同样，8b也完全烷基化了5‘-AGTCAGA-3’序列的G，并且在纳摩尔浓度下具有效率。为了更好地了解这些缀合物烷基化DNA的结构，研究了非自互补双十核苷酸ODNl和ODN2的烷基化。HPLC和ESMS分析表明，这两种odn与8a和8b的反应都能有效和选择性地偶联嘌呤碱基的N3烷基化。

Sequence-specific DNA alkylations have been a topic of much current interest due to their significant potential in molecular biology and human medicine. We developed general method to synthesize novel conjugates between segment A of Duocarmycin A (Du) and N-methylimidazole (lm)-N-methylpyrrole (Py) hairpin polyamides and their DNA alkylation. The conjugates PyPyPygammalmPyPyDu (8a) and JmPyPygammalmPyPyDu (8b) were designed to alkylate the target sequences (A/T)G(A/T)_2N(A/G) and (A/T)G(A/T)CN(A/G), respectively, according to Dervan's ring-pairing rule. High resolution denaturing gel electrophoresis indicated that 8a exclusively alkylated the A of the 5'-TGTAAAA-3' within a -400 bp DNA fragment Similarly, alkylation by 8b occurred exclusively at the G of the 5'-AGTCAGA-3' sequence with efficiency at nanomolar concentration. In order to better understand the structure of the alkylated DNA by these conjugates, the alkylation of non-self complementary duplex decanucleotides, ODNl and ODN2, were investigated. HPLC and ESMS analyses of the reaction of these ODNs with 8a and 8b demonstrated that both conjugates efficiently and selectively alkylate N3 of the purine bases of their target sequence.