IDENTIFICATION OF A BIOLOGICALLY SIGNIFICANT DNA-BINDING PEPTIDE MOTIF BY USE OF A RANDOM PHAGE DISPLAY LIBRARY

A peptide library approach was used to identify peptides that could bi nd to different DNA structures. A 23-mer random peptide library was di splayed in the context of the pIII protein of M13 filamentous phage. D ouble-stranded (ds) oligodeoxyribonucleotides (oligos) were immobilize d in 96-well plates using either chemical conjugation or a biotinavidi n linking method. Individual phage clones capable of binding to immobi lized oligos were selected from the phage library. Using a plaque dilu tion assay for rapid screening of binding preferences, four groups of oligo-binding (OB) phage were tentatively identified as showing prefer ence for: (1) single-stranded (ss) oligos irrespective of sequence; (2 ) ds oligos irrespective of sequence; (3) sequence-specific binding to ss oligos; and (4) weak non-specific binding to all types of oligos t ested. A quantitative solution-phase competition assay was used to con firm the ability of certain phage to discriminate ss from ds oligos. A consensus motif, FGRA, was found in those phage clones that preferent ially bound ss oligos; this motif has previously been noted in the bin ding domains of several ribonucleoproteins and ss DNA-binding proteins . Peptides based on the FGRA motif, but not scrambled controls, were a ble to inhibit the binding of appropriate phage clones or of Escherich ia coli ss DNA-binding protein to oligos. This suggests that amino aci d sequences that are capable of affecting biologically significant pro tein-DNA interactions can be identified from random peptide libraries using phage display techniques.