Development of the New Recognition Molecules of DNA Sequences Based on the GC- and AT-Selective Ligands

近年来，由于治疗应用的巨大潜力，开发特异性抑制基因表达的方法学已成为人们的主要兴趣。我们希望针对靶DNA序列开发新的配体。此外，通过对新配体与靶DNA之间的结合进行结构分析，将为序列选择性配体的设计提供有用的信息。在概念机制中，配体结合的主要过程包括从水溶液到DNA小槽的疏水转移。在这一步中，配体的形状与沟槽的曲率成等螺旋，是影响结合的重要因素。与已被证明具有螺旋状构象的低聚四氢呋喃化合物类似，具有长烷基链的双四氢呋喃化合物具有月牙形的稳定构象。MM计算表明，它们可能会很好地适应次要凹槽。为了验证这一假说，我们合成了以二氨基双四氢呋喃为公共骨架的各种新分子。引入氨基是为了使化合物溶于水并获得一定的静电结合力。综上所述，我们发现了基于二氨基双四氢呋喃骨架的新的DNA结合分子。从UV熔融曲线、乙锭置换实验和ITC测定的基础研究中，RR8与高亲和力的GC对的选择结合亲和力变得明显。也清楚地表明，氨基的立体化学和烷基链长对DNA结合起主要作用。DNase I的足迹分析表明，在这里研究的序列中，GCGC位点是RR8最匹配的位点。虽然RR8的详细结合模式尚不清楚，但新结合基序的GC偏好将为设计新的富含GC序列的结合分子提供有用的信息。

Development of methodology for specific inhibition of gene expression has become of major interest recently, because of great potential of therapeutic application. We expected to develope new ligands toward target DNA sequences. Furthermore, useful information for design of sequence selective ligands would be obtained by structural analysis of binding between the new ligands and the target DNA.In the conceptual mechanism, a primary process of the ligand-binding includes a hydrophobic transfer from an aqueous solution to the DNA minor groove. At this step, the shape of the ligand isohelical with the curvature of the groove is an important factor of the binding. Similar to oligo-tetrahydrofuran compounds that have been shown to take a helix-like conformation, bisTHF compounds with long alkyl chains take a crescent shape as the stable conformation. An MM calculation has indicated that they might fit well in the minor groove. In order to check this hypothesis, we synthesized a variety of new molecules with a diamino-bisTHF structure as the common skeleton. The amino groups were introduced to make a compound soluble in water and to gain some electrostatic binding forces.In conclusion, we have discovered new DNA-binding molecules based on a diamino-bisTHF skeleton. From the fundamental investigation with UV melting curve, ethidium displacement assay and the ITC measurement, selective binding affinity of RR8 to GC pairs with high affinity has become apparent. It has been also clearly shown that both the stereochemistry of the amino groups and the alkyl chain length play major roles for the DNA binding. Footprinting analyses with DNase I has revealed that a GCGC site is the most matched site for RR8 in the sequence studied here. Although the detailed binding mode of RR8 is not yet clear, GC-preference of the new binding motif will provide useful information for the design of new binding molecules for GC-rich sequence.