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

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

Structural Evaluation of Tandem Hairpin Pyrrole–Imidazole Polyamides Recognizing Human Telomeres

A polyamide containing <i>N</i>-methylpyrrole (Py) and <i>N</i>-methylimidazole (Im), designated PIPA, binds with high affinity and specificity to specific nucleotide sequences in the minor groove of double-helical DNA. Based on a recent report of the synthesis of PIPA for telomere visualization, the present paper focused on the size of the connecting part (hinge region) of two PIPA segments of the tandem hairpin PIPA, Dab­(Im-Im-Py)-Py-Py-Py-Im-[Hinge]-Dab­(Im-Im-Py)-Py-Py-Py-Im-βAla-NH­(CH₂)₃N­(CH₃)-(CH₂)₃NH-[Dye]. The present paper also describes the characterization of binding by measuring the thermal melting temperature and surface plasmon resonance and by specific staining of telomeres (TTAGGG)­n in human cells. Microheterogeneity was also investigated by high-resolution mass spectrometry. We found that the optimal compound as the hinge segment for telomere staining was [-NH­(C₂H₄O)₂(C₂H₄)­CO-] with tetramethylrhodamine as the fluorescent dye