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

Abstract

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)n-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 stain-ing 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.

Cyclic Naphthalene Diimide Dimer with a Strengthened Ability to Stabilize Dimeric G‐Quadruplex

“…According to the results (Table 1; Figure S18 and S19, Supporting Information), 2-4 and 5 showedasimilarly high affinity towards binding to as ingle G-quadruplex( over 2 10 6 m À1 ), and the cNDI-dimers' affinity were slightly highert han 5.H owever, recognizing dimeric G-quadruplexes, 2-4 exhibited ad ecreasedb inding affinity (around 3 10 5 m À1 ), which was lower than that of 5 towards dimeric G-quadruplex. [38] Although 3 showedaslightly stronger affinity towardsG -quadruplex than 2 and 4,t he difference was not significant.Ina ddition, for both 2-4 and 5,the energy changes towardsr ecognizing HP27 were quite small, and no clear binding affinity fitting curve could be obtained, which further indicated the enhanced ability of cNDI derivatives to discriminate the G-quadruplex and duplex DNA ( Figure S13, Supporting Information). [35,37] Another possible reason was that the bigger structure of cNDI-dimer mayd ecrease its accessibilityt od imericGquadruplex.…”

“…[35,37] Another possible reason was that the bigger structure of cNDI-dimer mayd ecrease its accessibilityt od imericGquadruplex. [38] Although 3 showedaslightly stronger affinity towardsG -quadruplex than 2 and 4,t he difference was not significant.Ina ddition, for both 2-4 and 5,the energy changes towardsr ecognizing HP27 were quite small, and no clear binding affinity fitting curve could be obtained, which further indicated the enhanced ability of cNDI derivatives to discriminate the G-quadruplex and duplex DNA ( Figure S13, Supporting Information).…”

Evaluation of alkylating pyrrole-imidazole polyamide conjugates by a novel method for high-throughput sequencer

“…These properties are seen in vitro, but experiments targeting genomic DNA in vivo, Chem-Seq 26 , enabled biological discussions at gene levels. 21,27,28 Here we investigated binding preferences of two PI polyamide conjugates using Bind-n-Seq analysis with a new MR (motif identification with a reference sequence) for the first time for alkylating conjugates as well as with the conventional MERMADE. In addition, their cytotoxicity against an A549 cell line was explored to expand the options for cancer treatment to targeting the RUNX family.…”

Interaction of fluorescently labeled pyrrole-imidazole polyamide probes with fixed and living murine and human cells

“…Future work could aim at improving the relative specificity of human probes for pericnetromeric DNA with respect to the rest of the genome. New tandem constructions via conjugation of two to four hairpin polyamides by a flexible linker may be envisioned, following the recent publications of Sugiyama's group [36,37]. This approach works well in fixed cells for imaging of telomere repeats, but for living cells, according to our preliminary data (data not shown), formation of aggregates is observed.…”