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

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。我们期望从具有巨大多样性的肽库中确定针对靶DNA序列的特异性肽配体。此外，通过对肽与靶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。我们已经报道了一种利用固态五肽库和靶结合磁珠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库和与靶DNA结合的磁珠，选择双链体DNA序列作为限制性内切酶Dra I的靶位点，将DNA结合的磁珠与库珠在磷酸盐缓冲液中混合。大多数相互作用的珠-珠复合物通过强外部磁体收集，然后最终在显微镜下分离。通过自动肽测序仪直接分析所选择的单珠-珠复合物，然后确定70个五肽序列。最常见的氨基酸是：第一个残基为苯丙氨酸，第二个残基为谷氨酰胺，第三个残基为甘氨酸、酪氨酸、色氨酸和苯丙氨酸，第四个和第五个残基均为异亮氨酸。有趣的是，疏水性氨基酸是最常见的。合成了12个这样确定的五肽，并定量分析了它们对双链DNA的亲和力。在所合成的五肽中，HMGII表现出对DNA的最高亲和力，高达Ks=2.6x10^6 M^I。因此，我们已经证明了组合技术可用于从固态文库中确定高亲和力的五肽配体，并且疏水性肽对双链体DNA具有高亲和力。

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.