Artificial DNA binding peptides recognizing separated sequences would expand varieties of the target genes for desirable transcriptional control. Here we demonstrated that polyarginine linker between two 3-zinc finger domains gives DNA binding selectivity to the separated target sequences. We created a six-zinc finger peptide, Sp1ZF6(Arg)8, by connecting two DNA binding domains of transcription factor Sp1 with a bulky and cationic polyarginine linker. The DNA binding properties to continuous and discontinuous target sequences were examined and compared to those of Sp1ZF6(Gly)10 containing a flexible and neutral polyglycine linker. The dissociation constants indicate that Sp1ZF6(Arg)8 has an obvious DNA binding preference to discontinuous target sequences but not Sp1ZF6(Gly)10. Footprinting analyses also showed that Sp1ZF6(Arg)8 binds properly only to the discontinuous target sites, while Sp1ZF6(Gly)10 does not distinguish them. The results provide helpful information for linker design of future zinc finger peptides to various states of DNA as gene expression regulators. (c) 2005 Elsevier Inc. All rights reserved.