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

由于具有巨大的治疗应用潜力，研究特异性抑制基因表达的方法已成为近年来研究的热点。我们期望开发出针对目标DNA序列的新配体。此外，通过对新配体与靶DNA结合的结构分析，可以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，配体的形状与凹槽的曲率等螺旋是结合的重要因素。与低聚四氢呋喃化合物呈螺旋状构象类似，具有长烷基链的双四氢呋喃化合物呈新月形作为稳定构象。MM计算表明，它们可以很好地配合小槽。为了验证这一假设，我们合成了多种以二氨基-双四氢呋喃结构为共同骨架的新分子。氨基的引入使化合物可溶于水，并获得一定的静电结合力。总之，我们发现了新的基于二氨基-双四氢呋喃骨架的dna结合分子。通过紫外熔融曲线、乙硫代置换法和ITC测定等基础研究，发现RR8对高亲和力的GC对具有选择性结合亲和力。还清楚地表明，氨基酸的立体化学性质和烷基链长度对DNA的结合起主要作用。DNase I的足迹分析显示，GCGC位点是研究序列中与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-preference将为设计新的富gc序列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.