Gene, 137 (1993) 13316 0 1993 Elsevier Science Publishers B.V. All rights reserved. 0378-l 119/93/$06.00 13 GENE 07419 Discovery of D-amino-acid-containing (Synthetic peptide library; antibody; streptavidin; mimotope) Kit S. Lama, Michal Leblb, Victor Krchnakb, Shelly Christine Cuzzocreab and Ken Wertmanb ligands with selectide technology Wadeb, Farid Abdul-Latif b, Ronald Fergusonb, zyxwvutsrqpo ‘Arizona Cancer Center, 1501 N. Campbell Avenue, and Department of Medicine, University of Arizona, Tucson, AZ 85724, USA; and bSelectide Corporation, 1580 E. Hanley Blvd., Tucson, AZ 85737, USA Received by J.W. Larrick/R.A. Houghton: 1 February 1993; Accepted: 22 March 1993; Received at publishers: 16 July 1993 SUMMARY When all L-amino acid (aa) random peptide libraries synthesized on solid-phase particles were screened (Selectide Technology), we identified several peptide ligands (YG_F_) that interacted specifically with an anti-&endorphin mono- clonal antibody (clone 3E7) (single-letter aa symbols; symbols ‘_’ indicate variable aa). Here, we report on the screening of three different D-aa-containing pentapeptide libraries (XxXxX, xXxXx and xxxxx, wherein X = L-aa, and x = o-aa) with the same antibody, in which several D-aa-containing ligands were identified. The binding affinities of many of these D- aa-containing ligands were as least two orders of magnitude lower than that of YGGFL, for which the Ki is 17.5 nM. INTRODUCTION Over the last few years, enormous effort has been given to the development of new methods to rapidly discover peptide ligands for target macromolecules. These meth- ods center around the idea of library screening, that is, to select specific peptide ligands from a large collection of random or semi-random peptide ligands. In general, there are two methods of constructing peptide libraries: (i) filamentous phage approach using molecular biology techniques (Cwirla et al., 1990; Devlin et al., 1990; Scott Correspondence to: Dr. K.S. Lam, Arizona Cancer Center, 1501 N. Campbell Avenue, Tucson, AZ 85724, USA. Tel. (l-602) 626-7925; Fax (l-602) 626-2284. Abbreviations: a, o-alanine; A, L-alanine; aa, amino acid(s); B, B-alanine; BEN, B-endorphin; BOP, benzotriazole-l-yl-oxy-tris-(dimethylamino)- phosphonium-hexa-fluorophosphate; DIEA, diisopropylethylamine; DMF, dimethylformamide; E, s-aminocaproic acid; EN, endorphin; f, o-phenylalanine; F, L-phenylalanine; Fmoc-aa, 9-fluorenyl- methoxycarbonyl amino acids; G, glycine; HOBt, N-hydroxybenzotria- zole; K,, inhibition binding constant; mAb, monoclonal antibody(ies); q, o-glutamine; Q, L-glutamine; t, D-threonine; w, D-tryptophan; W, L- tryptophan; x, all 18 o-aa plus glycine without cysteine; X, all 18 L-aa plus glycine without cysteine; y, o-tyrosine; Y, L-tyrosine; _, variable aa. et al., 1990), and (ii) synthetic peptide approach using solid phase peptide synthesis (Geysen et al., 1986; 1987; Fodor et al., 1991; Furka et al., 1991; Houghten et al., 1991; 1992; Lam et al., 1991; 1992; 1993; Lam and Lebl, 1992). Both methods have successfully proven to be useful in discovering peptide ligands specific for various target macromolecules: streptavidin (Devlin et al., 1990; Lam et al., 1991; Lam and Lebl, 1992), avidin (Lam and Lebl, 1992), mAb (Geysen et al., 1986; 1987; Cwirla et al., 1990; Scott et al., 1990; Houghten et al., 1991; 1992; Lam et al., 1991; 1992; 1993), concanavalin A (Oldenberg et al., 1992; Scott et al., 1992), opiate receptor (Houghten et al., 1992), and peptides with antibacterial activities (Houghten et al., 1991). The major limitation of the filamentous phage technique, however, is that the library has to consist of only genetically encoded natural aa since a biological system is used, whereas in the synthetic peptide library approach, unnatural aa can easily be incorporated into the constructs of the library. We described the use of a ‘split-synthesis’ approach to synthesize a random peptide library such that each bead expresses a single peptide entity. We then successfully demonstrated that using an enzyme-linked immunoassay technique, we were able to