A series of hairpin pyrrole-imidazole polyamide-fluorescein conjugates were synthesized and assayed for cellular localization. Thirteen cell lines, representing 11 human cancers, one human transformed kidney cell line, and one murine leukemia cell line, were treated with 5 M polyamide-fluorescein conjugates for 10 -14 h, then imaged by confocal laser scanning microscopy. A conjugate containing a ␤-alanine residue at the C terminus of the polyamide moiety showed no nuclear localization, whereas an analogous compound lacking the ␤-alanine residue was strongly localized in the nuclei of all cell lines tested. The localization profiles of several other conjugates suggest that pyrrole-imidazole sequence and content, dye choice and position, linker composition, and molecular weight are determinants of nuclear localization. The attachment of fluorescein to the C terminus of a hairpin polyamide results in an Ϸ10-fold reduction in DNA-binding affinity, with no loss of binding specificity with reference to mismatch binding sites.

A pivotal step forward in chemical approaches to controlling gene expression is the development of sequence-specific DNA-binding molecules that can enter live cells and traffic to nuclei unaided. DNA-binding polyamides are a class of programmable, sequence-specific small molecules that have been shown to influence a wide variety of protein-DNA interactions. We have synthesized over 100 polyamide-fluorophore conjugates and assayed their nuclear uptake profiles in 13 mammalian cell lines. The compiled dataset, comprising 1300 entries, establishes a benchmark for the nuclear localization of polyamide-dye conjugates. Compounds in this series were chosen to provide systematic variation in several structural variables, including dye composition and placement, molecular weight, charge, ordering of the aromatic and aliphatic amino-acid building blocks and overall shape. Nuclear uptake does not appear to be correlated with polyamide molecular weight or with the number of imidazole residues, although the positions of imidazole residues affect nuclear access properties significantly. Generally negative determinants for nuclear access include the presence of a beta-Ala-tail residue and the lack of a cationic alkyl amine moiety, whereas the presence of an acetylated 2,4-diaminobutyric acid-turn is a positive factor for nuclear localization. We discuss implications of these data on the design of polyamide-dye conjugates for use in biological systems.

Typical eukaryotic transcriptional activators are composed of distinct functional domains, including a DNA binding domain and an activating domain. Artificial transcription factors have been designed wherein the DNA binding domain is a minor groove DNA binding hairpin polyamide linked by a flexible tether to short activating peptides, typically 16-20 residues in size. In this study, the linker between the polyamide and the peptide was altered in an incremental fashion using rigid oligoproline "molecular rulers" in the 18-45 A length range. We find that there is an optimal linker length which separates the DNA and the activation region for transcription activation.