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)_<i>n</i>-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)_<i>n</i>-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