Selection of new sequence-selective unnatural peptides binding to double-stranded deoxyribonucleic acids (dsDNA) by means of a gel-retardation experiment for library analysis

Abstract

Previously, we developed a methodology for the solid-phase screening of peptide libraries for interaction with double-stranded deoxyribonucleic acids (dsDNA). In the search for new and more-potent DNA ligands, we investigated the strategy of solution-phase screening of chemical libraries consisting of unnatural oligopeptides. After synthesis of the selected amino acid building blocks, libraries were constructed with the general structure Ac-Arg-Ual-Sar-X-1-X-2-X-3-Arg-NH2, where X represents each of twelve unnatural or natural amino acids. Optimization of the sequence of binding peptides was performed with an iterative deconvolution procedure. Selection of interacting peptides was carried out in solution by means of gel-retardation experiments, starting with libraries of 144 compounds. A 14-base-pair double-stranded DNA fragment was chosen as the target. After several cycles of synthesis and screening of libraries and individual peptides, an oligopeptide was selected with an apparent dissociation constant of 9(.)10(-5)M, as determined by gel-retardation experiments. This peptide was studied by NMR spectroscopy. A certain degree of conformational pre-organization of the peptides was shown by temperature-dependent circular-dichroism experiments. Finally, DNase-I-footprinting studies indicated a preferential interaction with a 6-base-pair mixed sequence 5'-CTGCAT-3'. This study demonstrates that gel-shift experiments can be used for the solution-phase screening of library mixtures of peptides against dsDNA. In general, this technique allows the selection of new sequence-selective dsDNA-interacting molecules. Furthermore, novel dsDNA-binding unnatural oligopeptides were developed with affinities in the 0.1 mu range.