A pyrrole-imidazole polyamide motif for recognition of eleven base pair sequences in the minor groove of DNA

A new upper limit of binding site size is defined for the 2:1 overlapped polyamide:DNA motif. Eight-ring polyamides composed of four-ring subunits containing pyrrole (Py) and imidazole (Im) amino acids linked by a central beta-alanine (beta) spacer (''4-beta-4 ligands'') were designed for recognition of eleven base pair sequences as antiparallel dimer (4-beta-4)(2) DNA complexes in the minor groove. The DNA binding properties of three polyamides, ImPyPyPy-beta-PyPyPyPy-beta-Dp, ImImPyPy-beta-PyPyPyPy-beta-Dp, and ImImImPy-beta-PyPyPyPy-beta-Dp, were analyzed by footprinting experiments on DNA fragments containing the respective match sites 5'-AGTAATTTACT-3', 5'-AGGTATTACCT-3' (Dp = dimethylaminopropylamide). Quantitative footprint titrations reveal that each polyamide binds its respective target site with subnanomolar affinity and 7-fold to over 30-fold specificity over double-base-pair mismatch sites. A 20-fold decrease in binding affinity is observed for placement of a side-by-side beta-beta pairing opposite G•C/C•G relative to placement opposite a A•T/T•A base pair. The use of side-by-side antiparallel beta-alanine residues as an A•T/T•A-specific DNA binding element provides a new pairing rule for polyamide design. Expanding the DNA binding site size targeted by pyrrole-imidazole polyamides represents an important step in the development of cell-permeable synthetic ligands for the control of gene-specific regulation