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

基于 GC 和 AT 选择性配体的新型 DNA 序列识别分子的开发

基本信息

批准号：
11672105
负责人：
SASAKI Shigeki
金额：
$ 2.3万
依托单位：
KYUSHU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11672105/
关键词：
COMBINATORIAL CHEMISTRY SOLID-STATE LIBRARY PEPTIDE LIBRARY DUPLEX DNA DNA-BINDING MOLECULES HYDROPHOBIC AMINO ACIDS DNA結合

项目摘要

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.

由于基因表达的特异性抑制在治疗上具有巨大的应用潜力，因此基因表达特异性抑制方法的研究成为近年来的热点。我们期望开发针对靶DNA序列的新配体。此外，通过对配体与靶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，在理论上，配体与靶DNA结合的主要过程包括从水溶液到DNA小沟的疏水转移。在这一步中，配体等螺旋的形状与凹槽的曲率是结合的重要因素。类似于已显示采取螺旋状构象的低聚四氢呋喃化合物，具有长烷基链的bisTHF化合物采取新月形形状作为稳定构象。MM计算表明，它们可能很好地适合小沟。为了验证这一假设，我们合成了多种具有二氨基-bisTHF结构作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DNA结合分子的基础上二氨基双THF骨架。从UV熔解曲线、乙锭置换试验和ITC测量的基础研究中，RR 8对具有高亲和力的GC对的选择性结合亲和力变得明显。它也已清楚地表明，氨基的立体化学和烷基链的长度发挥主要作用的DNA结合。DNA酶I足迹分析表明，GCGC位点是RR 8在这里研究的序列中最匹配的位点。虽然RR 8的详细结合模式尚不清楚，但新结合基序的GC偏好性将为设计新的富含GC序列的结合分子提供有用的信息。