22 research outputs found
Synthesis of Pyrrole–Imidazole Polyamide <i>seco</i>-1-Chloromethyl-5-hydroxy-1,2-dihydro‑3<i>H</i>‑benz[<i>e</i>]indole Conjugates with a Vinyl Linker Recognizing a 7 bp DNA Sequence
Convergent synthetic routes for <i>N</i>-methylpyrrole
(P) and <i>N</i>-methylimidazole (I) <i>seco</i>-1-chloromethyl-5-hydroxy-1,2-dihydro-3<i>H</i>-benz[<i>e</i>]indole (CBI) conjugates with a vinyl linker were developed.
New hairpin polyamide–<i>seco</i>-CBI conjugates,
compounds <b>16</b>–<b>19</b>, were synthesized,
and their DNA sequence-specific alkylating activities were evaluated
via high-resolution denaturing gel electrophoresis and high-performance
liquid chromatography (HPLC) product analysis. The new synthetic route
for PI conjugates with a vinyl linker consisted of the introduction
of a vinylpyrrole unit (<b>8</b>–<b>11</b>) into
the C terminal of a PI polyamide synthesized by (fluorenylmethoxy)carbonyl
solid-phase peptide synthesis (SPPS), followed by liquid-phase coupling
with <i>seco</i>-CBI. The yield of the conjugates was significantly
improved compared with that of the method reported previously, which
allows us to synthesize various substituted conjugates containing
a vinyl linker. Conjugates <b>16</b>–<b>19</b> were
designed to investigate the substituent effect of the vinyl linker,
and conjugate <b>16</b><i><b>S</b></i> was synthesized
to evaluate the reactivity between racemic and <i>S</i> enantiomers
of the <i>seco</i>-CBI derivative. The results of high-resolution
denaturing gel electrophoresis using 208 bp DNA fragments indicated
that alkylation by compounds <b>16</b> and <b>17</b>,
in which the H of the vinyl linker of compound <b>16</b> was
replaced with F, occurred predominantly at the A of the 5′-TTTGTCA-3′
sequence at nanomolar concentrations. In clear contrast, compounds <b>18</b> and <b>19</b>, which were methyl or Br derivatives
of compound <b>16</b>, did not exhibit any DNA alkylating activity.
Moreover, HPLC product analysis using synthetic oligonucleotides demonstrated
that alkylation occurred between the N3 of the adenine of the oligomer
and the cyclopropane ring of <b>16</b><i><b>S</b></i>. Density functional calculation of substituted vinylpyrrole <i>seco</i>-CBI units indicated that methyl and Br substituents
led to a significantly distorted geometry of the vinyl group with
the pyrrole ring compared with H and F derivatives. Molecular modeling
studies offered the additional information that steric hindrance reduced
the DNA alkylating activity of these derivatives
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
Results of real-time quantification of PCR GFP () and β-actin () mRNAs in HCT116 cells treated with polyamides A–C
<p><b>Copyright information:</b></p><p>Taken from "Alkylation of template strand of coding region causes effective gene silencing"</p><p>Nucleic Acids Research 2006;34(4):1189-1195.</p><p>Published online 25 Feb 2006</p><p>PMCID:PMC1383623.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> To evaluate the amount of transcribed GFP mRNA, TaqMan real-time PCR was performed with the 7300 Real-Time PCR System. The amount of GFP and β-actin mRNAs in the HCT116 cells were measured using pairs of primers with the TaqMan probes for each gene. The amount of mRNAs in HCT116 cells treated with 100 nM polyamides for 24 h was calculated with reference to the cells treated with 0.1% DMF (Control), set as 100%. Compared with the control, HCT116 cells treated with polyamide C showed significantly decreased GFP mRNA expression. The cells which the GFP vectors were not transfected (Mock) did not express GFP mRNA. Error bars, standard deviation of the means of triplicate samples. * < 0.05 by unpaired Student's -test compared with control
Thermally induced strand cleavage of 5′-Texas Red-labeled DNA fragments of GFP former ( and ) and latter ( and ) sequences of GFP alkylated by polyamides A, B and C
<p><b>Copyright information:</b></p><p>Taken from "Alkylation of template strand of coding region causes effective gene silencing"</p><p>Nucleic Acids Research 2006;34(4):1189-1195.</p><p>Published online 25 Feb 2006</p><p>PMCID:PMC1383623.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> Results in the sequence of complementary strand (a–c) and coding strand (d-f) are displayed. Lanes 1–4: 100, 50, 25, 12.5 nM of A; lanes 5–8: 100, 50, 25, 12.5 nM of B, lanes 9–12: 100, 50, 25, 12.5 nM of C and lane 13: DNA control. Lanes G, C, T and A contain Sanger-sequencing products ( and ). Sequences containing alkylation sites are represented. Mismatched sequences are indicated by gray letters
Sequence-Selective Single-Molecule Alkylation with a Pyrrole–Imidazole Polyamide Visualized in a DNA Nanoscaffold
We demonstrate a novel strategy for visualizing sequence-selective
alkylation of target double-stranded DNA (dsDNA) using a synthetic
pyrrole–imidazole (PI) polyamide in a designed DNA origami
scaffold. Doubly functionalized PI polyamide was designed by introduction
of an alkylating agent 1-(chloromethyl)-5-hydroxy-1,2-dihydro-3<i>H</i>-benz[<i>e</i>]indole (<i>seco</i>-CBI)
and biotin for sequence-selective alkylation at the target sequence
and subsequent streptavidin labeling, respectively. Selective alkylation
of the target site in the substrate DNA was observed by analysis using
sequencing gel electrophoresis. For the single-molecule observation
of the alkylation by functionalized PI polyamide using atomic force
microscopy (AFM), the target position in the dsDNA (∼200 base
pairs) was alkylated and then visualized by labeling with streptavidin.
Newly designed DNA origami scaffold named “five-well DNA frame”
carrying five different dsDNA sequences in its cavities was used for
the detailed analysis of the sequence-selectivity and alkylation.
The 64-mer dsDNAs were introduced to five individual wells, in which
target sequence AGTXCCA/TGGYACT (XY = AT, TA, GC, CG) was employed
as fully matched (X = G) and one-base mismatched (X = A, T, C) sequences.
The fully matched sequence was alkylated with 88% selectivity over
other mismatched sequences. In addition, the PI polyamide failed to
attach to the target sequence lacking the alkylation site after washing
and streptavidin treatment. Therefore, the PI polyamide discriminated
the one mismatched nucleotide at the single-molecule level, and alkylation
anchored the PI polyamide to the target dsDNA
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
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
NanoScript: A Nanoparticle-Based Artificial Transcription Factor for Effective Gene Regulation
Transcription factor (TF) proteins are master regulators of transcriptional activity and gene expression. TF-based gene regulation is a promising approach for many biological applications; however, several limitations hinder the full potential of TFs. Herein, we developed an artificial, nanoparticle-based transcription factor, termed NanoScript, which is designed to mimic the structure and function of TFs. NanoScript was constructed by tethering functional peptides and small molecules called synthetic transcription factors, which mimic the individual TF domains, onto gold nanoparticles. We demonstrate that NanoScript localizes within the nucleus and initiates transcription of a reporter plasmid by over 15-fold. Moreover, NanoScript can effectively transcribe targeted genes on endogenous DNA in a nonviral manner. Because NanoScript is a functional replica of TF proteins and a tunable gene-regulating platform, it has great potential for various stem cell applications