Influence of structural variation on nuclear localization of DNA-binding polyamide-fluorophore conjugates

Abstract

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.

Completion of a programmable DNA-binding small molecule library

“…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.…”

“…29,30 Cell lines were trypsinized (Mediatech) for 5 min at 37°C, centrifuged for 10 min at 4°C at 100 × g, and resuspended in fresh medium to a concentration of 1.33 × …”

“…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.…”