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 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.

Completion of a programmable DNA-binding small molecule library

“…A chiral 2,4-diaminobutyric acid improves affi nity while retaining specifi city for A•T and T•A. 29 The increased binding affi nity can be attributed to electrostatic interactions between the negatively charged DNA backbone and the positively charged free amine on the turn. Furthermore, the chiral turn allows stereochemical control, enforcing the N→C polyamide alignment with respect to the 5'→3' orientation of the adjacent DNA strand.…”

“…Furthermore, the chiral turn allows stereochemical control, enforcing the N→C polyamide alignment with respect to the 5'→3' orientation of the adjacent DNA strand. 29,30 The microstructure of DNA is sequence-dependent, and polyamides composed of Py and Im rings are overly curved compared to the DNA minor groove. Aliphatic β-alanine residues have been incorporated at internal positions to reset the register between contiguous aromatic ring pairings.…”

“…28 The acetylated turn linkage used in the design of compounds 1, 3, and 5 has been shown to improve nuclear localization in some human cancer cell lines. 29,30 Polyamides 1-5 were synthesized on oxime resin according to published manual solidphase synthesis protocols. 31,32 Fluorescein and isophthalic acid conjugations, as well as the subsequent deprotection and acetylation steps, were performed according to published methods.…”

“…31,32 Fluorescein and isophthalic acid conjugations, as well as the subsequent deprotection and acetylation steps, were performed according to published methods. 26,[28][29][30] Polyamides 6 and 7 were synthesized as mismatch control compounds for cell culture experiments and have been previously characterized. 28 Binding isotherms are shown, and θ norm values were calculated according to published methods.…”

“…20,22,25,26 Within the framework of the pairing rules, covalent linkages between two antiparallel polyamide strands result in several possible structures, including the hairpin, cycle, Hpin, and U-pin binding motifs. [27][28][29][30] These linked structures show improved affi nity and specifi city when compared with the unlinked dimers. The eight-ring hairpin polyamide provides a good compromise between synthetic ease (linear vs. branched oligomers) and molecular recognition properties.…”