Nuclear Localization and Gene Expression Modulation by a Fluorescent Sequence-Selective p-Anisyl-benzimidazolecarboxamido Imidazole-Pyrrole Polyamide

Abstract

Synthetic pyrrole (P)-imidazole (I) containing polyamides can target predetermined DNA sequences and modulate gene expression by interfering with transcription factor binding. We have previously shown that rationally designed polyamides targeting the inverted CCAAT box 2 (ICB2) of the topoisomerase IIα (topo IIα) promoter can inhibit binding of transcription factor NF-Y, re-inducing expression of the enzyme in confluent cells. Here, the A/T recognizing fluorophore, p-anisylbenzimidazolecarboxamido (Hx) was incorporated into the hybrid polyamide HxIP, which fluoresces upon binding to DNA, providing an intrinsic probe to monitor cellular uptake. HxIP targets the 5'-TACGAT-3' sequence of the 5' flank of ICB2 with high affinity and sequence specificity, eliciting an ICB2-selective inhibition/displacement of NF-Y. HxIP is readily taken up by NIH3T3 and A549 cells, and detected in the nucleus within minutes. Exposure to the polyamide at confluence resulted in a dose-dependent upregulation of topo IIα expression and enhanced formation of etoposide-induced DNA strand breaks.

Modulation of topoisomerase IIα expression and chemosensitivity through targeted inhibition of NF-Y:DNA binding by a diamino p-anisyl-benzimidazole (Hx) polyamide

“…DNase I footprinting studies on the biologically relevant topo IIα promoter (Figure 4) reveal that HxI*P 2 binds to the 5′-TACGAT-3′ target sequence of the 5′-flank of the ICB2 with weaker affinity (3 μM) than the monoamino HxIP 1 (1 μM, [28]) despite showing greater affinity in the biophysical studies. HxIP* 3 displayed a 2-fold enhancement of binding affinity relative to polyamide 1 , generating a footprint evident at 0.5 μM, which is in strong agreement with the SPR results.…”

“…The DNase I footprinting reactions were performed as described by Kiakos et al [28] and resolved on a 10% denaturing polyacrylamide-urea gel (National Diagnostics) by electrophoresis for 3 h at 1650 V (55°C) in 1× TBE buffer. The gel was then transferred onto Whatman 3MM paper, dried and exposed overnight to Fuji medical X-ray film to visualise the radioactive signal.…”

“…Electrophoretic mobility shift assay (EMSA) experiments were conducted as outlined by Kiakos et al [28] using nuclear protein extracts from NIH3T3 fibroblast cells and the ICB2-containing oligonucleotide; 5′-GGCAAGCTAC G ATTGG TTCTTCTGGACG-3′ (sense); 5′-CGTCCAGAAGAACCAATCGTAGCTTGCC-3′ (antisense). Oligonucleotides containing a mutated ICB2 were used as specific competitors, with the wild-type ICB motif replaced by AAACC and GGTTT in sense and antisense oligonucleotides respectively.…”

“…Most recently, we reported the synthesis and biological activity of a novel polyamide incorporating the p -anisylbenzimidazole (Hx) DNA recognition element [28], [29]. Designed to enhance polyamide-DNA binding and cellular uptake, the Hx moiety exhibits intrinsic fluorescence upon binding DNA enabling the direct visualisation of polyamide nuclear localisation.…”

“…Hx-polyamide HxIP ( 1 , Figure 2A) designed to target the 5′-flanking sequence 5′-TACGAT-3′ of the ICB2 (Figure 1), binds to DNA with high affinity and sequence selectivity, and disrupts the NF-Y:ICB2 interaction resulting in the upregulation of topo IIα expression at confluence. HxIP pre-treatment enhanced etoposide-induced DNA damage, providing further evidence that sequence specific polyamides can re-sensitise confluence-arrested cancer cells to topo IIα poisons [28]. …”