Random Search of DNA Recognition Molecules by the Use of the New Combinatorial Technology

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。我们期望从具有巨大多样性的多肽文库中确定针对靶DNA序列的特异性多肽配体。此外，通过对多肽与靶DNA结合的结构分析，将为序列选择性配体的设计提供有用的信息。我们已经报道了一种利用固态五肽文库和靶标共轭磁珠在组合技术应用中进行随机搜索的新方法。在本研究中，我们使用相同的固态五肽文库和与目的DNA偶联的磁珠，选择双链DNA序列作为限制性内切酶Dra I的靶点。大多数相互作用的珠子-珠子复合体被一个强大的外磁体收集，然后在显微镜下分离。用全自动多肽测序仪直接分析所选择的单个珠粒-珠粒复合体，然后测定了70个五肽序列。氨基酸组成以苯丙氨酸最多，谷氨酰胺次之，甘氨酸、酪氨酸、色氨酸、苯丙氨酸次之，第四和第五位均含有异亮氨酸。有趣的是，疏水性氨基酸是最常被测定的。合成了12个五肽，并对它们与双链DNA的亲和力进行了定量分析。在合成的五肽中，FQGII与DNA的亲和力最高，Ks=2.6×10~(-6)M~(-1)。因此，我们证明了组合技术对于从固态文库中确定高亲和力的五肽配体是有用的，并且疏水性多肽对双链DNA具有很高的亲和力。

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 determine specific peptide ligands toward target DNA sequences from peptide library with enormous diversity. Furthermore, useful information for design of sequence selective ligands would be obtained by structural analysis of binding between peptides and the target DNA.We have already reported a new method for random search in the application of combinatorial technology by utilizing solid-state pentapeptide library and target-conjugated magnetic beads. In this study, we used the same solid-state pentapeptide library and the magnetic beads conjugating the target DNA.The duplex DNA sequence was selected as the target site of the restriction enzyme Dra I.We mixed the DNA-conjugated magnetic beads with the library beads in a phosphate buffer. Most interacting bead-bead complexes were collected by a strong outer magnet, then finally separated under a microscope. The selected single bead-bead complex was directly analyzed by an automated peptide sequencer, then seventy sequences of pentapeptides were determined. Following amino acids were found most frequently ; phenylalanine at the first residue, glutamine at the second, glycine, tyrosine, tryptophane, and phenylalanine at the third, and isoleucine at both the fourth and the fifth residues. Interestingly, hydrophobic amino acids were most frequently determined. Twelve pentapeptides thus determined were synthesized and their affinity toward duplex DNAs were quantitatively analyzed. Among the synthesized pentapeptides, FQGII exhibited the highest affinity to DNA as much as Ks=2.6x10^6 M^I.Thus, we have demonstrated that the combinatorial technology is useful to determine high affinity pentapeptide ligands from the solid-state library, and that hydrophobic peptides have high affinity to the duplex DNA.