Tandem hairpin motif for recognition in the minor groove of DNA by pyrrole- Imidazole polyamides

six-ring hairpin polyamides linked tail-to-turn by a five-carbon tether rec ognize a predetermined Ii-base-pair (bp) site in the minor groove of DNA. P olyamide subunits, containing three pyrrole (Py) or imidazole (Im) aromatic amino acids covalently linked by a turn-specific gamma-aminobutyric acid ( gamma-turn) residue, form six-ring hairpin structures that recognize design ated five-base-pair sequences. Replacement of the gamma-turn residue with ( R)-2,4,-diaminobutyric acid [(R)(H2N)gamma] provides for enhanced hairpin-D NA-binding affinity and sequence specificty. In order to extend the targeta ble binding-site size of the hairpin motif, two tandem hairpin polyamides, ImPyPy-(R)[ImPyPy-(R)(H2N)gamma PyPyPy beta](HN)gamma PyPyPy beta Dp (1) an d ImPyPy-(R)[ImPyPy(R)(H2N)gamma PyPyPy delta](HN)gamma PyPyPy beta Dp (2), were designed such that the carboxy tail of one six-ring hairpin is covale ntly tethered to the (R)(H2N)gamma-turn of the second through beta-alanine (beta) or 5-aminovaleric acid (delta), respectively. The DNA-binding affini ty of each polyamide was characterized by quantitative footprint titration experiments on DNA fragments containing 10-, 11-, or 12-bp match and mismat ch sequences. The parent six-ring hairpin ImPyPy(R)(H2N)gamma-PyPyPy-beta-D p binds to a 5-bp 5'-TGTTA-3' half site with an equilibrium association con stant (K-a)= 5 x 10(9)M(-1) and 100-fold specificity versus a 5'-TGTCA-3' m ismatch site. The tandem-hairpin polyamide 2, linked by valeric acid, binds the Il-bp site 5'- TGTTATTGTTA-3' (individual 6-ring hairpin target sites underlined) with K-a greater than or equal to 1 x 10(12)M(-1) and greater t han or equal to 4500-fold specificity versus the double mismatch sequence 5 '-TGTCATTGTCA-3', The 10-bp and 12-bp sites 5'-TGTTATGTTA-3' and 5'-TGTTATT TGTTA-3' are bound with at least 70 and 1000-fold reduced affinity, respect ively. beta- linked polyamide 1 binds to both the 10- and 11-bp sites with K-a = 2 x 10(10)M(-1) and to the 12-bp site with K-a = 9 x 10(8)M(-1). The results presented here identify structure elements that expand polyamide-bi nding-site size by linking previously described hairpin recognition units. Remarkably, a simple aliphatic 5-carbon tether is sufficient to provide inc reased binding affinity without comprimising hairpin sequence-selectivity.