7 research outputs found
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)<sub><i>n</i></sub>-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)<sub><i>n</i></sub>-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