Improved nuclear localization of DNA-binding polyamides

Abstract

Regulation of endogenous genes by DNA-binding polyamides requires effective nuclear localization. Previous work employing confocal microscopy to study uptake of fluorophore-labeled polyamides has demonstrated the difficulty of predicting a priori the nuclear uptake of a given polyamide. The data suggest that dye identity influences uptake sufficiently such that a dye-conjugate cannot be used as a proxy for unlabeled analogs. Polyamides capable of nuclear localization unaided by fluorescent dyes are desirable due to size and other limitations of fluorophores. Recently, a polyamide-fluorescein conjugate targeted to the hypoxia response element (HRE) was found to inhibit vascular endothelial growth factor (VEGF) expression in cultured HeLa cells. The current study uses inhibition of VEGF expression as a biological read-out for effective nuclear localization of HRE-targeted polyamides. We synthesized a focused library of non-fluorescent, HRE-targeted polyamides in which the C-terminus ‘tail’ has been systematically varied. Members of this library bind the HRE with affinities comparable or superior to that of the fluorescein-labeled analog. Although most library members demonstrate modest or no biological activity, two non-fluorescent polyamides are reported with activity rivaling that of the previously reported fluorescein-labeled polyamide. We also show evidence that promoter occupancy by HIF-1, the transcription factor that binds the HRE, is inhibited by HRE-targeted polyamides.

Completion of a programmable DNA-binding small molecule library

“…[23][24][25][26] While several different binding motifs have been explored, the hairpin motif provides excellent affi nity and specifi city for DNA and can be accessed through a facile synthetic route. 23 In the hairpin motif, a γ-aminobutyric acid (γ) covalently links the carboxylic terminus of one polyamide with the amino terminus of the other.…”

“…This class of polyamide conjugates has been shown to exhibit improved nuclear localization. 26 Polyamides 2 and 3 contain a chiral 2,4-diaminobutyric acid turn linkage that improves the binding affi nity over the achiral variant without loss of sequence specifi city. 27 Polyamides 4 and 5 contain a chiral 3,4-diaminobutyric acid turn linkage that has been shown to increase the DNA binding affi nity further with some, but not all, polyamide cores.…”

“…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.…”

“…23,24 NMR and X-ray crystallography studies reveal that the crescent-shaped polyamide sideby-side dimer binds B-form DNA, a remarkable example of shape-selective recognition of the deep minor groove of DNA. 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.…”

Development of a molecule-recognized promoter DNA sequence for inhibition of HER2 expression

“…18 In addition, this compound has a different character from other genesilencing tools, such as siRNA or antisense oligo nucleotides, because penetration in the living cells, cytosol import and nuclear transport of PI polyamide occur without any delivery system and may not be influenced by any catabolic enzymes or metabolic enzymes, such as nucleases and P450 enzymes, even in animals. [19][20][21][22] These findings highlight the advantages of PI polyamide as a suitable candidate compound for pre-transcriptional HER2 gene silencing. An earlier publication has also reported that PI polyamide compounds showed specific binding at the Ets-binding site of the HER2/neu promoter region and inhibition of HER2/neu promoter-driven transcription measured in a cell-free system using nuclear extract from a human breast cancer cell line, SKBR-3.…”