225 research outputs found

    Cyclic pyrrole-imidazole polyamides targeted to the androgen response element

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    Hairpin pyrrole−imidazole (Py-Im) polyamides are a class of cell-permeable DNA-binding small molecules that can disrupt transcription factor−DNA binding and regulate endogenous gene expression. The covalent linkage of antiparallel Py-Im ring pairs with an γ-amino acid turn unit affords the classical hairpin Py-Im polyamide structure. Closing the hairpin with a second turn unit yields a cyclic polyamide, a lesser-studied architecture mainly attributable to synthetic inaccessibility. We have applied our methodology for solution-phase polyamide synthesis to cyclic polyamides with an improved high-yield cyclization step. Cyclic 8-ring Py-Im polyamides 1−3 target the DNA sequence 5′-WGWWCW-3′, which corresponds to the androgen response element (ARE) bound by the androgen receptor transcription factor to modulate gene expression. We find that cyclic Py-Im polyamides 1−3 bind DNA with exceptionally high affinities and regulate the expression of AR target genes in cell culture studies, from which we infer that the cycle is cell permeable

    Programming multiple protein patterns on a single DNA nanostructure

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    The ability to create assemblies of proteins with spacing on the nanometer scale has important implications for proteomics, biodetection, and self-assembly. Structural DNA nanotechnology has led to the creation of a variety of nanostructures which should be capable of serving as an addressable template for the creation of complex molecular assemblies. The goal of such systems is to be able to position proteins or other components in distinct patterns with precise spacing. These systems take advantage of the well-defined structure and spacing of DNA and use these properties to act as a template for secondary components in a bottom-up approach toward self-assembly. Previous work in this area has primarily focused on the use of chemical or structural modifications of the DNA template in order to attach or recruit proteins or nanoparticles. We have recently shown that a single polyamide-biotin conjugate is capable of binding to a DX array made from two tiles without any modification of the target DNA

    Inhibition of RNA polymerase II transcription in human cells by synthetic DNA-binding ligands

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    Sequence-specific DNA-binding small molecules that can permeate human cells potentially could regulate transcription of specific genes. Multiple cellular DNA-binding transcription factors are required by HIV type 1 for RNA synthesis. Two pyrrole-imidazole polyamides were designed to bind DNA sequences immediately adjacent to binding sites for the transcription factors Ets-l, lymphoid-enhancer binding factor 1, and TATA-box binding protein. These synthetic ligands specifically inhibit DNA-binding of each transcription factor and HIV type 1 transcription in cell-free assays. When used in combination, the polyamides inhibit virus replication by >99% in isolated human peripheral blood lymphocytes, with no detectable cell toxicity, The ability of small molecules to target predetermined DNA sequences located within RNA polymerase II promoters suggests a general approach for regulation of gene expression, as well as a mechanism for the inhibition of viral replication

    Addressing single molecules on DNA nanostructures

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    The synthesis of devices and materials from molecular components is a major goal of nanotechnology. Although many such molecular components have been demonstrated previously,1–3 the ability to combine these components into designed architectures containing significant complexity remains a challenge. By using the hybridization properties of DNA and Watson–Crick base pairing, it has been possible to create well-defined DNA architectures of increasing complexity.4 The structure of an assembled DNA complex is directly and uniquely determined by the sequence of the DNA bases, which can be designed and manipulated. These methods provide a versatile and programmable way to control the structure and architecture of DNA nanostructures

    Addressing single molecules on DNA nanostructures

    Get PDF
    The synthesis of devices and materials from molecular components is a major goal of nanotechnology. Although many such molecular components have been demonstrated previously,1–3 the ability to combine these components into designed architectures containing significant complexity remains a challenge. By using the hybridization properties of DNA and Watson–Crick base pairing, it has been possible to create well-defined DNA architectures of increasing complexity.4 The structure of an assembled DNA complex is directly and uniquely determined by the sequence of the DNA bases, which can be designed and manipulated. These methods provide a versatile and programmable way to control the structure and architecture of DNA nanostructures

    Single-dose pharmacokinetic and toxicity analysis of pyrrole–imidazole polyamides in mice

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    Purpose: Pyrrole–imidazole (Py-Im) polyamides are programmable, sequence-specific DNA minor groove–binding ligands. Previous work in cell culture has shown that various polyamides can be used to modulate the transcriptional programs of oncogenic transcription factors. In this study, two hairpin polyamides with demonstrated activity against androgen receptor signaling in cell culture were administered to mice to characterize their pharmacokinetic properties. Methods: Py-Im polyamides were administered intravenously by tail vein injection. Plasma, urine, and fecal samples were collected over a 24-h period. Liver, kidney, and lung samples were collected postmortem. Concentrations of the administered polyamide in the plasma, excretion, and tissue samples were measured using LC/MS/MS. The biodistribution data were analyzed by both non-compartmental and compartmental pharmacokinetic models. Animal toxicity experiments were also performed by monitoring weight loss after a single subcutaneous (SC) injection of either polyamide. Results: The biodistribution profiles of both compounds exhibited rapid localization to the liver, kidneys, and lungs upon injection. Plasma distribution of the two compounds showed distinct differences in the rate of clearance, the volume of distribution, and the AUCs. These two compounds also have markedly different toxicities after SC injection in mice. Conclusions: The variations in pharmacokinetics and toxicity in vivo stem from a minor chemical modification that is also correlated with differing potency in cell culture. The results obtained in this study could provide a structural basis for further improvement of polyamide activity both in cell culture and in animal models

    Replication stress by Py–Im polyamides induces a non-canonical ATR-dependent checkpoint response

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    Pyrrole–imidazole polyamides targeted to the androgen response element were cytotoxic in multiple cell lines, independent of intact androgen receptor signaling. Polyamide treatment induced accumulation of S-phase cells and of PCNA replication/repair foci. Activation of a cell cycle checkpoint response was evidenced by autophosphorylation of ATR, the S-phase checkpoint kinase, and by recruitment of ATR and the ATR activators RPA, 9-1-1, and Rad17 to chromatin. Surprisingly, ATR activation was accompanied by only a slight increase in single-stranded DNA, and the ATR targets RPA2 and Chk1, a cell cycle checkpoint kinase, were not phosphorylated. However, ATR activation resulted in phosphorylation of the replicative helicase subunit MCM2, an ATR effector. Polyamide treatment also induced accumulation of monoubiquitinated FANCD2, which is recruited to stalled replication forks and interacts transiently with phospho-MCM2. This suggests that polyamides induce replication stress that ATR can counteract independently of Chk1 and that the FA/BRCA pathway may also be involved in the response to polyamides. In biochemical assays, polyamides inhibit DNA helicases, providing a plausible mechanism for S-phase inhibition

    Tumor Repression of VCaP Xenografts by a Pyrrole-Imidazole Polyamide

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    Pyrrole-imidazole (Py-Im) polyamides are high affinity DNA-binding small molecules that can inhibit protein-DNA interactions. In VCaP cells, a human prostate cancer cell line overexpressing both AR and the TMPRSS2-ERG gene fusion, an androgen response element (ARE)-targeted Py-Im polyamide significantly downregulates AR driven gene expression. Polyamide exposure to VCaP cells reduced proliferation without causing DNA damage. Py-Im polyamide treatment also reduced tumor growth in a VCaP mouse xenograft model. In addition to the effects on AR regulated transcription, RNA-seq analysis revealed inhibition of topoisomerase-DNA binding as a potential mechanism that contributes to the antitumor effects of polyamides in cell culture and in xenografts. These studies support the therapeutic potential of Py-Im polyamides to target multiple aspects of transcriptional regulation in prostate cancers without genotoxic stress
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