103 research outputs found
Alkylation of template strand of coding region causes effective gene silencing
We recently developed a new type of pyrrole (Py)–imidazole (Im) polyamide–tetrahydrocyclopropabenzindolone (CBI) conjugate with an indole linker as a stable sequence-specific alkylating agent. In this study, we investigated the gene silencing activities of polyamides A, B and C, which selectively alkylate specific sequences in the promoter region, non-coding strand and coding strand, respectively, of the green fluorescent protein (GFP) gene. GFP vectors were transfected into human colon carcinoma cells (HCT116), and the cells were treated with 100 nM of the polyamides for 24 h. Fluorescence microscopy indicated that a significant reduction of GFP fluorescence was only observed in the cells that were treated with polyamide C. In clear contrast, polyamides A and B did not show such activity. Moreover, real-time PCR demonstrated selective reduction of the expression of GFP mRNA following treatment with polyamide C. These results suggest that alkylating Py–Im polyamides that target the coding strand represent a novel approach for sequence-specific gene silencing
Pyrrole-imidazole hairpin polyamides with high affinity at 5′–CGCG–3′ DNA sequence; influence of cytosine methylation on binding
To investigate the binding of 5′–CpG–3′ sequences by small molecules, two pyrrole (Py)–imidazole (Im) hairpin polyamides, PyImPyIm–γ–PyImPyIm–β–Dp (1) and PyIm–β–Im–γ–PyIm–β–Im–β–Dp (2), which recognize the sequence 5′–CGCG–3′, were synthesized. The binding affinities of the 5′–CGCG–3′ sequence to the Py–Im hairpin polyamides were measured by surface plasmon resonance (SPR) analysis. SPR data revealed that dissociation equilibrium constants (Kd) of polyamides 1 and 2 were 1.1 (± 0.3) × 10–6 M and 1.7 (± 0.4) × 10–8 M, respectively. Polyamide 2 possesses great binding affinity for this sequence, 65-fold higher than polyamide 1. Moreover, when all cytosines in 5′–CpGpCpG–3′ were replaced with 5-methylcytosines (mCs), the Kd value of polyamide 2 increased to 5.8 (± 0.7) × 10–9 (M), which indicated about 3-fold higher binding than the unmethylated 5′–CGCG–3′ sequence. These results suggest that polyamide 2 would be suitable to target CpG-rich sequences in the genome
Strong and Specific Recognition of CAG/CTG Repeat DNA (5'-dWGCWGCW-3') by a Cyclic Pyrrole-Imidazole Polyamide
Abnormally expanded CAG/CTG repeat DNA sequences lead to a variety of neurological diseases, such as Huntington's disease. Here, we synthesized a cyclic pyrrole-imidazole polyamide (cPIP), which can bind to the minor groove of the CAG/CTG DNA sequence. The double-stranded DNA melting temperature (Tm ) and surface plasmon resonance assays revealed the high binding affinity of the cPIP. In addition, next-generation sequencing showed that the cPIP had high specificity for its target DNA sequence
Evaluation of alkylating pyrrole-imidazole polyamide conjugates by a novel method for high-throughput sequencer
N-Methylpyrrole-N-methylimidazole (PI) polyamides are a class of DNA minor groove binders with DNA sequence-specificity. DNA-alkylating PI polyamide conjugates are attractive candidates as anticancer drugs acting through DNA damage and its subsequent inhibition of cell proliferation. One example is a chlorambucil-PI polyamide conjugate targeting the runt-related transcription factor (RUNX) family. RUNX1 has pro-oncogenic properties in acute myeloid leukemia, and recently the chlorambucil-PI polyamide conjugate was demonstrated to have anticancer effects. Herein, we apply another DNA-alkylating agent, seco-CBI, to target the consensus sequence of the RUNX family. Two types of CBI conjugates were prepared and their binding properties were characterized by Bind-n-Seq analysis using a high-throughput sequencer. The sequencing data were analyzed by two methods, MERMADE and our new MR (motif identification with a reference sequence), and the resultant binding motif logos were as predicted from the pairing rules proposed by Dervan et al. This is the first report to employ the MR method on alkylating PI polyamide conjugates. Moreover, cytotoxicity of conjugates 3 and 4 against a human non-small cell lung cancer, A549, were examined to show promising IC[50]s of 120 nm and 63 nm, respectively. These findings suggest seco-CBI-PI polyamide conjugates are candidates for oncological therapy
A synthetic small molecule for rapid induction of multiple pluripotency genes in mouse embryonic fibroblasts.
Cellular reprogramming involves profound alterations in genome-wide gene expression that is precisely controlled by a hypothetical epigenetic code. Small molecules have been shown to artificially induce epigenetic modifications in a sequence independent manner. Recently, we showed that specific DNA binding hairpin pyrrole-imidazole polyamides (PIPs) could be conjugated with chromatin modifying histone deacetylase inhibitors like SAHA to epigenetically activate certain pluripotent genes in mouse fibroblasts. In our steadfast progress to improve the efficiency of SAHA-PIPs, we identified a novel compound termed, δ that could dramatically induce the endogenous expression of Oct-3/4 and Nanog. Genome-wide gene analysis suggests that in just 24 h and at nM concentration, δ induced multiple pluripotency-associated genes including Rex1 and Cdh1 by more than ten-fold. δ treated MEFs also rapidly overcame the rate-limiting step of epithelial transition in cellular reprogramming by switching "[Formula: see text]" the complex transcriptional gene network
Advancing small-molecule-based chemical biology with next-generation sequencing technologies
Next-generation-sequencing (NGS) technologies enable us to obtain extensive information by deciphering millions of individual DNA sequencing reactions simultaneously. The new DNA-sequencing strategies exceed their precursors in output by many orders of magnitude, resulting in a quantitative increase in valuable sequence information that could be harnessed for qualitative analysis. Sequencing on this scale has facilitated significant advances in diverse disciplines, ranging from the discovery, design, and evaluation of many small molecules and relevant biological mechanisms to maturation of personalized therapies. NGS technologies that have recently become affordable allow us to gain in-depth insight into small-molecule-triggered biological phenomena and empower researchers to develop advanced versions of small molecules. In this review we focus on the overlooked implications of NGS technologies in chemical biology, with a special emphasis on small-molecule development and screening
A Synthetic Transcriptional Activator of Genes Associated with the Retina in Human Dermal Fibroblasts.
Small molecules capable of modulating epigenetic signatures can activate the transcription of tissue-restricted genes in a totally unrelated cell type and have potential use in epigenetic therapy. To provide an example for an initial approach, we report here on one synthetic small-molecule compound-termed "SAHA-PIP X"-from our library of conjugates. This compound triggered histone acetylation accompanied by the transcription of retinal-tissue-related genes in human dermal fibroblasts (HDFs)
DNA Interstrand Crosslinks by H-pin Polyamide (S)-seco-CBI Conjugates
Although DNA interstrand crosslinking (ICL) agents are widely used as antitumor drugs, DNA sequence-specific ICL agents are quite rare. In this study, H-pin imidazole-pyrrole polyamide 1-(chloromethyl)-2, 3-dihydro-1H-benzo[e]indol-5-ol (seco-CBI) conjugates that produce sequence-specific DNA ICLs were designed and synthesized. Conjugates with H-pin polyamide and seco-CBI moieties were constructed to recognize a 7 bp DNA sequence, and their reactivity and selectivity in DNA alkylation were evaluated by using high-resolution denaturing gel electrophoresis and sequence-specific plasmid cleavage. One conjugate (6), which contained a chiral (S)-seco-CBI, exhibited greater sequence-specific ICL activity toward the target DNA sequence and was cytotoxic to a cancer cell line. Molecular modeling studies indicated that the greater activity of 6 resulted from the relative orientation of the cyclopropane group in the (S)-CBI unit
W-derived BAC probes as a new tool for identification of the W chromosome and its aberrations in Bombyx mori
We isolated four W chromosome-derived bacterial artificial chromosome (W-BAC) clones from Bombyx mori BAC libraries by the polymerase chain reaction and used them as probes for fluorescence in situ hybridization (FISH) on chromosome preparations from B. mori females. All four W-BAC probes surprisingly highlighted the whole wild-type W sex chromosome and also identified the entire original W-chromosomal region in W chromosome-autosome translocation mutants. This is the first successful identification of a single chromosome by means of BAC-FISH in species with holokinetic chromosomes. Genomic in situ hybridization (GISH) by using female-derived genomic probes highlighted the W chromosome in a similar chromosome-painting manner. Besides the W, hybridization signals of W-BAC probes also occurred in telomeric and/or subtelomeric regions of the autosomes. These signals coincided well with those of female genomic probes except one additional GISH signal that was observed in a large heterochromatin block of one autosome pair. Our results support the opinion that the B. mori W chromosome accumulated transposable elements and other repetitive sequences that also occur, but scattered, elsewhere in the respective genome. Edited by: E.R. Schmid
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)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 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
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