| Budget Amount *help |
¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 1998: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1997: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Research Abstract |
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.
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