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.
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.
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.
A G-quadruplex (quadruplex) is a nucleic acid secondary structure adopted by guanine-rich sequences and is considered to be relevant to various pharmacological and biological contexts. Although a number of researchers have endeavored to discover and develop quadruplex-interactive molecules, poor ligand designability originating from topological similarity of the skeleton of diverse quadruplexes has remained a bottleneck for gaining specificity for individual quadruplexes. This work reports on hybrid molecules that were constructed with dual DNA-binding components, a cyclic imidazole/lysine polyamide (cIKP), and a hairpin pyrrole/imidazole polyamide (hPIP), with the aim toward specific quadruplex targeting by reading out the local duplex DNA sequence adjacent to designated quadruplexes in the genome. By means of circular dichroism (CD), fluorescence resonance energy transfer (FRET), surface plasmon resonance (SPR), and NMR techniques, we showed the dual and simultaneous recognition of the respective segment via hybrid molecules, and the synergistic and mutual effect of each binding component that was appropriately linked on higher binding affinity and modest sequence specificity. Monitoring quadruplex and duplex imino protons of the quadruplex/duplex motif titrated with hybrid molecules clearly revealed distinct features of the binding of hybrid molecules to the respective segments upon their simultaneous recognition. A series of the systematic and detailed binding assays described here showed that the concept of simultaneous recognition of quadruplex and its proximal duplex by hybrid molecules constructed with the dual DNA-binding components may provide a new strategy for ligand design, enabling targeting of a large variety of designated quadruplexes at specific genome locations.
Introducing novel building blocks to solid-phase peptide synthesis, we readily synthesized long-chain hairpin pyrrole-imidazole (PI) polyamide-chlorambucil conjugates 3 and 4 via the introduction of an amino group into a GABA (γ-turn) contained in 3, to target CAG/CTG repeat sequences, which are associated with various hereditary disorders. A high-resolution denaturing polyacrylamide sequencing gel revealed sequence-specific alkylation both strands at the N3 of adenines or guanines in CAG/CTG repeats by conjugates 3 and 4, with 11bp recognition. In vitro transcription assays using conjugate 4 revealed that specific alkylation inhibited the progression of RNA polymerase at the alkylating sites. Chiral substitution of the γ-turn with an amino group resulted in higher binding affinity observed in SPR assays. These assays suggest that conjugates 4 with 11bp recognition has the potential to cause specific DNA damage and transcriptional inhibition at the alkylating sites.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.