Ultra Bright LED Light Injection Calibration System for MINOS

We describe here a proposal for a light injection calibration system for the MINOS detectors based on ultra bright blue LEDs as the light source. We have shown that these LEDs are bright enough to span over two orders of magnitude in light intensity, commensurate with that expected in a single scintillator strip in the MINOS neutrino detectors.Comment: 9 pages, 13 figures, Submitted to NI

In vivo imaging of pyrrole-imidazole polyamides with positron emission tomography

The biodistribution profiles in mice of two pyrrole-imidazole polyamides were determined by PET. Pyrrole-imidazole polyamides are a class of small molecules that can be programmed to bind a broad repertoire of DNA sequences, disrupt transcription factor-DNA interfaces, and modulate gene expression pathways in cell culture experiments. The 18F-radiolabeled polyamides were prepared by oxime ligation between 4-[18F]-fluorobenzaldehyde and a hydroxylamine moiety at the polyamide C terminus. Small animal PET imaging of radiolabeled polyamides administered to mice revealed distinct differences in the biodistribution of a 5-ring β-linked polyamide versus an 8-ring hairpin, which exhibited better overall bioavailability. In vivo imaging of pyrrole-imidazole polyamides by PET is a minimum first step toward the translation of polyamide-based gene regulation from cell culture to small animal studies

Footprinting with MPE•Fe(II). Complementary-strand analyses of distamycin- and actinomycin-binding sites on heterogeneous DNA

We recently reported a direct technique for determining the binding sites of small molecules on naturally occurring heterogeneous DNA (Van Dyke et al. 1982). Methidiumpropyl-EDTA·Fe(II) (MPE·Fe[II]) (Hertzberg and Dervan 1982) cleaves double-helical DNA with low sequence-specificity (Van Dyke et al. 1982). Using a combination of MPE·Fe(II) partial cleavage of drug-protected DNA fragments and Maxam-Gilbert sequencing methods, we determined the drug-protected sites on one strand of a double-helical fragment from pBR322 for the intercalator actinomycin D (Goldberg et al. 1962; Muller and Crothers 1968; Wells and Larson 1970; Sobell 1973; Krugh 1981; Patel et al. 1981; Takusagawa et al. 1982) and the minor-groove binders netropsin and distamycin A (Luck et al. 1974; Wartell et al. 1974; Zimmer 1975; Berman et al. 1979; Krylov et al. 1979). Netropsin and distamycin A gave identical DNA-cleavage inhibition patterns or footprints in regions rich in dA·dT base pairs. Actinomycin D afforded a completely different footprint..

Programming multiple protein patterns on a single DNA nanostructure

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

Repression of DNA-binding dependent glucocorticoid receptor-mediated gene expression

The glucocorticoid receptor (GR) affects the transcription of genes involved in diverse processes, including energy metabolism and the immune response, through DNA-binding dependent and independent mechanisms. The DNA-binding dependent mechanism occurs by direct binding of GR to glucocorticoid response elements (GREs) at regulatory regions of target genes. The DNA-binding independent mechanism involves binding of GR to transcription factors and coactivators that, in turn, contact DNA. A small molecule that competes with GR for binding to GREs could be expected to affect the DNA-dependent pathway selectively by interfering with the protein-DNA interface. We show that a DNA-binding polyamide that targets the consensus GRE sequence binds the glucocorticoid-induced zipper (GILZ) GRE, inhibits expression of GILZ and several other known GR target genes, and reduces GR occupancy at the GILZ promoter. Genome-wide expression analysis of the effects of this polyamide on a set of glucocorticoid-induced and -repressed genes could help to elucidate the mechanism of GR regulation for these genes

Modulation of NF-κB-dependent gene transcription using programmable DNA minor groove binders

Nuclear factor κB (NF-κB) is a transcription factor that regulates various aspects of immune response, cell death, and differentiation as well as cancer. In this study we introduce the Py-Im polyamide 1 that binds preferentially to the sequences 5′-WGGWWW-3′ and 5′GGGWWW-3′. The compound is capable of binding to κB sites and reducing the expression of various NF-κB–driven genes including IL6 and IL8 by qRT-PCR. Chromatin immunoprecipitation experiments demonstrate a reduction of p65 occupancy within the proximal promoters of those genes. Genome-wide expression analysis by RNA-seq compares the DNA-binding polyamide with the well-characterized NF-κB inhibitor PS1145, identifies overlaps and differences in affected gene groups, and shows that both affect comparable numbers of TNF-α–inducible genes. Inhibition of NF-κB DNA binding via direct displacement of the transcription factor is a potential alternative to the existing antagonists

Expanding the Repertoire of Natural Product-Inspired Ring Pairs for Molecular Recognition of DNA

A furan amino acid, inspired by the recently discovered proximicin natural products, was incorporated into the scaffold of a DNA-binding hairpin polyamide. While unpaired oligomers of 2,4-disubstituted furan amino acids show poor DNA-binding activity, furan (Fn) carboxamides paired with N-methylpyrrole (Py) and N-methylimidazole (Im) rings demonstrate excellent stabilization of duplex DNA as well as discrimination of noncognate sequences, consistent with function as a Py mimic according to the Py/Im polyamide pairing rules

Microwave Assisted Synthesis of Py-Im Polyamides

Microwave synthesis was utilized to rapidly build Py-Im polyamides in high yields and purity using Boc-protection chemistry on Kaiser oxime resin. A representative polyamide targeting the 5′-WGWWCW-3′ (W = A or T) subset of the consensus Androgen and Glucocorticoid Response Elements was synthesized in 56% yield after 20 linear steps and HPLC purification. It was confirmed by Mosher amide derivatization of the polyamide that a chiral α-amino acid does not racemize after several additional coupling steps

Toward artificial developmental regulators

A polyamide-peptide conjugate is designed which recruits sequence specifically the developmental regulator Exd to a cognate DNA site. In particular, an eight-ring hairpin polyamide (Im-Im-Py(C3H6NHR)-Py-gamma-Im-Py-Py-Py-beta-Dp) with a heptapeptide (R = Ac-Phe-Tyr-Pro-Trp-Met-Lys-Gly-) attached on a central ring was shown to induce cooperative binding of the Drosophila Hox protein cofactor Exd with a Kd of 4.4 nM in vitro, an order of magnitude more efficient than the natural Hox protein partner Ubx. The conjugate joins two sequence specific domains, one for DNA and one for the protein. This small molecule thus serves as a cooperative protein-DNA dimerizer, which mimics the natural Hox family of developmental regulators