Design of Gene Therapeutic Agent Possessing Cooperative DNA Alkylating Ability

序列特异的DNA烷基化反应因其在分子生物学和人类医学中的巨大潜力而成为当前研究的热点。我们开发了一种新的多卡霉素A(DU)A链段与N-甲基咪唑(Lm)-N-甲基吡咯(Py)发夹状聚酰胺偶联物的合成方法及其DNA烷基化反应。根据Dervan的环配对规则，设计了两个偶联物，分别用于烷基化目标序列(A/T)G(A/T)_2N(A/G)和(A/T)G(A/T)CN(A/G)。高分辨变性凝胶电泳法表明，8a仅在-400bp的DNA片段中对5‘-TGTAAAA-3’的A发生烷基化作用，8b的烷基化作用仅发生在5‘-AGTCAGA-3’序列的G处，在纳摩尔浓度范围内有效。为了更好地了解这些偶联物的烷基化DNA的结构，我们研究了非自互补双链十核苷酸ODN1和ODN2的烷基化反应。对这些ODN与8a和8b反应的高效液相色谱和ESMS分析表明，这两种偶联物都能有效和选择性地使其靶序列的嘌呤碱基的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.