G‑Quadruplex Induction by the Hairpin Pyrrole–Imidazole Polyamide Dimer

The G-quadruplex (G4) is one type of higher-order structure of nucleic acids and is thought to play important roles in various biological events such as regulation of transcription and inhibition of DNA replication. Pyrrole–imidazole polyamides (PIPs) are programmable small molecules that can sequence-specifically bind with high affinity to the minor groove of double-stranded DNA (dsDNA). Herein, we designed head-to-head hairpin PIP dimers and their target dsDNA in a model G4-forming sequence. Using an electrophoresis mobility shift assay and transcription arrest assay, we found that PIP dimers could induce the structural change to G4 DNA from dsDNA through the recognition by one PIP dimer molecule of two duplex-binding sites flanking both ends of the G4-forming sequence. This induction ability was dependent on linker length. This is the first study to induce G4 formation using PIPs, which are known to be dsDNA binders. The results reported here suggest that selective G4 induction in native sequences may be achieved with PIP dimers by applying the same design strategy

Specific Alkylation of Human Telomere Repeat Sequences by a Tandem-Hairpin Motif of Pyrrole–Imidazole Polyamides with Indole-<i>seco</i>-CBI

We designed and synthesized a tandem-hairpin motif of pyrrole (P)imidazole (I) polyamide 1-(chloromethyl)-5-hydroxy-1,2-dihydro-3<i>H</i>-benz­[e]­indole (<i>seco</i>-CBI) conjugates (<b>1</b>) that targets the human telomere repeat sequence 5′-d­(CCCTAA)_<i>n</i>-3′. As a control, conjugate <b>2</b> (hairpin PI polyamide with <i>seco</i>-CBI), which also targets the human telomere repeat sequence, was synthesized. High-resolution denaturing polyacrylamide gel electrophoresis (PAGE) using 5′ Texas Red-labeled 219-bp DNA fragments revealed the outstandingly high sequence selectivity of <b>1</b>, with no mismatch alkylation. Furthermore, an evaluation performed in human cancer cell lines demonstrated that conjugate <b>1</b> has low cytotoxicity compared with conjugate <b>2</b>. In addition, a cell-staining analysis indicated that conjugate <b>1</b> induced apoptosis moderately by DNA damage. This study demonstrated that conjugate <b>1</b> can be used as an effective alkylator for telomere repeat sequences or as an apoptotic inducer

Development of a New Method for Synthesis of Tandem Hairpin Pyrrole–Imidazole Polyamide Probes Targeting Human Telomeres

Pyrrole–imidazole (PI) polyamides bind to the minor groove of DNA in a sequence-specific manner without causing denaturation of DNA. To visualize telomeres specifically, tandem hairpin PI polyamides conjugated with a fluorescent dye have been synthesized, but the study of telomeres using these PI polyamides has not been reported because of difficulties synthesizing these tandem hairpin PI polyamides. To synthesize tandem hairpin PI polyamides more easily, we have developed new PI polyamide fragments and have used them as units in Fmoc solid-phase peptide synthesis. Using this new method, we synthesized four fluorescent polyamide probes for the human telomeric repeat TTAGGG, and we examined the binding affinities and specificities of the tandem hairpin PI polyamides, the UV–vis absorption and fluorescence spectra of the fluorescent polyamide probes, and telomere staining in mouse MC12 and human HeLa cells. The polyamides synthesized using the new method successfully targeted to human and mouse telomeres under mild conditions and allow easier labeling of telomeres in the cells while maintaining the telomere structure. Using the fluorescent polyamides, we demonstrated that the telomere length at a single telomere level is related to the abundance of TRF1 protein, a shelterin complex component in the telomere

Creation of a Synthetic Ligand for Mitochondrial DNA Sequence Recognition and Promoter-Specific Transcription Suppression

Synthetic ligands capable of recognizing the specific DNA sequences inside human mitochondria and modulating gene transcription are in increasing demand because of the surge in evidence linking mitochondrial genome and diseases. In the work described herein, we created a new type of mitochondria-specific synthetic ligand, termed MITO-PIPs, by conjugating a mitochondria-penetrating peptide with pyrrole-imidazole polyamides (PIPs). The designed MITO-PIPs showed specific localization inside mitochondria in HeLa cells and recognized the target DNA in a sequence-specific manner. Furthermore, MITO-PIPs that inhibit the binding of mitochondrial transcription factor A to the light-strand promoter (LSP) also triggered targeted transcriptional suppression. The tunability of PIPs’ properties suggests the potential of the MITO-PIPs as potent modulators of not only mitochondrial gene transcription but also its DNA mutations

Biomimetic Artificial Epigenetic Code for Targeted Acetylation of Histones

While the central role of locus-specific acetylation of histone proteins in eukaryotic gene expression is well established, the availability of designer tools to regulate acetylation at particular nucleosome sites remains limited. Here, we develop a unique strategy to introduce acetylation by constructing a bifunctional molecule designated Bi-PIP. Bi-PIP has a P300/CBP-selective bromodomain inhibitor (Bi) as a P300/CBP recruiter and a pyrrole–imidazole polyamide (PIP) as a sequence-selective DNA binder. Biochemical assays verified that Bi-PIPs recruit P300 to the nucleosomes having their target DNA sequences and extensively accelerate acetylation. Bi-PIPs also activated transcription of genes that have corresponding cognate DNA sequences inside living cells. Our results demonstrate that Bi-PIPs could act as a synthetic programmable histone code of acetylation, which emulates the bromodomain-mediated natural propagation system of histone acetylation to activate gene expression in a sequence-selective manner

Targeting 24 bp within Telomere Repeat Sequences with Tandem Tetramer Pyrrole–Imidazole Polyamide Probes

Synthetic molecules that bind sequence-specifically to DNA have been developed for varied biological applications, including anticancer activity, regulation of gene expression, and visualization of specific genomic regions. Increasing the number of base pairs targeted by synthetic molecules strengthens their sequence specificity. Our group has been working on the development of pyrrole–imidazole polyamides that bind to the minor groove of DNA in a sequence-specific manner without causing denaturation. Recently, we reported a simple synthetic method of fluorescent tandem dimer polyamide probes composed of two hairpin moieties with a linking hinge, which bound to 12 bp in human telomeric repeats (5′-(TTAGGG)_<i>n</i>-3′) and could be used to specifically visualize telomeres in chemically fixed cells under mild conditions. We also performed structural optimization and extension of the target base pairs to allow more specific staining of telomeres. In the present study, we synthesized tandem tetramer polyamides composed of four hairpin moieties, targeting 24 bp in telomeric repeats, the longest reported binding site for synthetic, non-nucleic-acid-based, sequence-specific DNA-binding molecules. The novel tandem tetramers bound with a nanomolar dissociation constant to 24 bp sequences made up of four telomeric repeats. Fluorescently labeled tandem tetramer polyamide probes could visualize human telomeres in chemically fixed cells with lower background signals than polyamide probes reported previously, suggesting that they had higher specificity for telomeres. Furthermore, high-throughput sequencing of human genomic DNA pulled down by the biotin-labeled tandem tetramer polyamide probe confirmed its effective binding to telomeric repeats in the complex chromatinized genome

Synthesis and Biological Properties of Highly Sequence-Specific-Alkylating <i>N</i>-Methylpyrrole–<i>N</i>-Methylimidazole Polyamide Conjugates

Four new alkylating <i>N</i>-methylpyrrole-<i>N</i>-methylimidazole (PI) polyamide conjugates (<b>1</b>–<b>4</b>) with seven-base-pair (bp) recognition ability were synthesized. Evaluation of their DNA-alkylating activity clearly showed accurate alkylation at match site­(s). The cytotoxicities of conjugates <b>1</b>–<b>4</b> were determined against six human cancer cell lines, and the effect of these conjugates on the expression levels of the whole human genome in A549 cells were also investigated. A few genes among the top 20 genes were commonly downregulated by each conjugate, which reflects their sequence specificity. Conversely, many of the top 10 genes were commonly upregulated, which may have been caused by alkylation damage to DNA. Moreover, the antitumor activities of the PI polyamide conjugates <b>2</b> and <b>3</b> were investigated using nude mice transplanted with DU145 or A549. The intravenous administration of each liposomal conjugate in water yielded tumor-suppressing effects specifically toward DU145 cells and not A549 cells, which was pertinent to cytotoxicity