In order to target specific DNA sequences ≥10 base pairs in size by minor groove binding ligands, a search for the optimal linker in dimers of hairpin polyamides was initiated. Two series of tandem polyamides ImPyIm-( R)[ImPyIm-(R) H2Nγ-PyPyPy-L] HNγ-PyPyPy-β-Dp ( 1a– e), where L represents a series of 4–8 carbon long aliphatic amino acid linkers, and ImPyIm-( R)[ImPyIm-(R) H2Nγ-PyPyPyIm-L] HNγ-PyPyPy-β-Dp ( 2a– e), where L represents a series of 2–6 carbon long aliphatic amino acid linkers, were synthesized and characterized by quantitative DNase I footprinting. β, γ and Dp represents β-alanine, γ-aminobutyric acid, and 3-(dimethylamino)propylamine, respectively. It was found that the five-carbon 5-aminovaleric acid (δ), is suitable to span one base-pair (bp) of DNA when incorporated into a tandem polyamide. ImPyIm-( R)[ImPyIm-(R) H2Nγ-PyPyPy-δ] HNγ-PyPyPy-β-Dp ( 1b) binds the 10 bp binding-site 5′-AGTGAAGTGA-3′ with equilibrium association constant K a=3.2×10 10 M −1 and ImPyIm-( R)[ImPyIm-(R) H2Nγ-PyPyPyIm-δ] HNγ-PyPyPy-β-Dp ( 2d) binds the 11 bp binding-site 5′-AGTGATAGTGA-3′ with K a=9.7×10 9 M −1. Tandem 1b also bind the 11 bp site but with lower affinity affording a 15-fold specificity for the shorter binding site. Replacing a methylene group in the amino acid linker with an oxygen atom to form tandem polyamide ImPyIm-( R)[ImPyIm-(R) H2Nγ-PyPyPy-E] HNγ-PyPyPy-β-Dp ( 4) where E represents the ether linker, resulted in that an 80-fold specificity for the 10 bp binding site over the 11 bp site.