Nuclear localization of pyrrole-imidazole polyamide-fluorescein conjugates in cell culture

Abstract

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 microM polyamide-fluorescein conjugates for 10-14 h, then imaged by confocal laser scanning microscopy. A conjugate containing a beta-alanine residue at the C terminus of the polyamide moiety showed no nuclear localization, whereas an analogous compound lacking the beta-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 approximately 10-fold reduction in DNA-binding affinity, with no loss of binding specificity with reference to mismatch binding sites.

Fig. 1.

Structures of compounds 1–22, which were synthesized for this work, and compound 23, which was purchased from Molecular Probes.

Fig. 2.

Cellular localization of polyamide–fluorescein conjugates. (Upper) Adherent MCF-7 cells were treated with compound 2 (Upper Left)or 3 (Upper Right) for 10–14 h at 5 μM. Compound 2 was excluded from the cells entirely, whereas compound 3 localized to the nucleus. (Bottom) Suspended Jurkat cells were similarly treated and show similar results. (Scale bar = 10 μm.)

Fig. 3.

Colocalization of polyamide 3 and Hoechst in live cells, imaged by using sequential single- and two-photon excitation. (Upper) MCF-7 cells were treated with the nuclear stain Hoechst dye 33342 (15 μM), compound 3 (5 μM), and the dead cell stain Sytox Orange (0.5 μM). Fluorescence signals from Hoechst (Upper Left, blue) and compound 3 (Upper Center, green) colocalize in cell nuclei. (Upper Right) Overlay of the visible light image (grayscale) and the Sytox Orange fluorescence image (red), indicating that the majority of cells are alive. (Lower) Jurkat cells were treated similarly and show similar results. (Scale bar = 10 μm.)

Fig. 4.

Uptake profile of compounds 1–22 in 13 cell lines. + +, Nuclear staining exceeds that of the medium; +, nuclear staining less than or equal to that of the medium, but still prominent; -, very little nuclear staining, with the most fluorescence seen in the cytoplasm and/or medium; --, no nuclear staining.

Fig. 5.

Quantitative DNase I footprinting titration. Compound 3 binds the 3′-TGGTCA-5′ site with K_a = 1.6 (±0.3) × 10⁹ M^-1. Compound 3 does not bind the single base pair mismatch sites shown at concentrations ≤200 nM.