Netropsin-poly(dA-dT) complex in solution: structure and dynamics of antibiotic-free base pair regions and those centered on bound netropsin.

Abstract

The biphasic duplex-to-strand transition for the netropsin-poly(dA-dT) complex, phosphate/drug mole ratio (P/D) = 50, has been investigated by high-resolution proton nuclear magnetic resonance (NMR) spectroscopy at the nonexchangeable base and sugar protons in 0.1 M cacodylate solution. The NMR spectral parameters monitor the structure and dynamics of thle opening of antibiotic-free base pair regions Netropsin is a basic oligopeptide isolated from Streptomyces netropsis which is composed of 1-methylpyrrole-2-carboxamide moieties linked to guanidinoacetamidino and amidinopropyl groups ( Fig. 1) (1). It exhibits antiviral, antifungal, and antibacterial activities and inhibits DNA and RNA tumor viruses in mammalian cells (2). Netropsin complexes with duplex DNA but does not complex with single-stranded DNA, double-stranded RNA, or DNA-RNA hybrids (3,4). DNA and RNA synthesis is inhibited following netropsin interaction with the template and/or primer nucleic acid duplex (4, 5). Netropsin neither intercalates nor unwinds the DNA double helix (4, 6). Studies on a series of synthetic polynucleotides demonstrate the presence of one major binding site with the netropsin molecule spanning from three to five base pairs (7-9). Netropsin complexes to synthetic DNAs containing dA-dT, dA-dBrU, dI-dC, and dI-dBrC base pairs but not to synthetic DNAs containing dG-dC base pairs (3, 4). This suggests that netropsin binds in the minor groove and that the 2-amino group of guanine inhibits complex formation (4). Models for the netropsin-DNA complex stress the importance of electrostatic interactions between the charged ends of the antibiotic and the backbone phosphates and the contributions from peptide-The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked NMR Spectra. Proton NMR spectra were recorded in the Fourier transform mode on a Bruker HX-360 NMR spectrometer interfaced to a Nicolet BNC-12 computer. The chemical shifts are referenced relative to internal standard, 2,2-dimethyl-2-silapentane-5-sulfonate (DSS). The temperature of the probe was calibrated to ±1 using ethyleneglycol.Ultraviolet-Visible Spectra. Optical studies were undertaken on samples (0.25 cm3 volume) placed in water-jacketed 1-mm path length cylindrical Helma cells with Teflon caps, and the UV-visible absorbance data were recorded on a Cary 118 C spectrometer. Automatic stepwise increases in temperature of 10/min followed by a dwell time of 4 min were maintained by a TP2 programmer attached to a RTE-8 Neslab circulating bath. An immersible microprobe (Bailey IT-1 implantable, 18 gauge) was put into the reference solution through a bore in the Teflon cap and the temperature was monitored by a digital thermometer (Bailey BAT-8). The nucleic acid 260-and 278-nm absorbance values were read directly on the digital readout of the Cary 118 C following zeroing of the baseline at 700 nm at each temperature.Abbreviations: NMR, nuclear magnetic resonance; P/D, phosphate/ dr...

Mutual interaction between adjacent dG . dC actinomycin binding sites and dA . dT netropsin binding sites on the self-complementary d(C-G-C-G-A-A-T-T-C-G-C-G) duplex in solution.

“…2). This conclusion is supported by earlier NMR investigations which demonstrated that netropsin at low binding ratios induces a conformational change in poly(dA-dT) which is propagated to adjacent antibiotic-free base pair regions (10). Actinomycin and Netropsin Complex.…”

Interaction of minor groove ligands to an AAATT/AATTT site: correlation of thermodynamic characterization and solution structure

“…At the ionic strength of these studies, the magnitude of Kb for the association of just one Dst ligand is characteristic of the binding of Net and Dst to sites containing all dA'dT base pairs (Marky et al, 1983(Marky et al, , 1985 Marky & Kupke, 1989; Marky, 1986;Breslauer et al, 1986Breslauer et al, , 1987 Marky & Breslauer, 1987b). On the basis of the available structures of Net-and Dst-DNA complexes with different sequences (Pelton & Wemmer, 1988, 1990aGeirstanger et al, 1993;Patel & Canuel, 1977;Patel, 1979Patel, , 1982, we suggest that hydrogen bonding contributions to the overall binding strength are similar. Net and the first Dst molecule of the 2:1 complex have similar overall binding constants.…”

May the Best Molecule Win: Competition ESI Mass Spectrometry

“…Analysis of competitive binding of compounds with a set of multiple, mixed sequence DNA sequences by ESI-MS is an attractive method that has been used in a number of applications. Initial tests were performed with minor groove binding compounds, netropsin [ 83 , 84 , 85 , 86 , 87 , 88 ] and DB75 [ 89 , 90 , 91 ], which are well-characterized. They are often used as standards in method development with DNA and minor groove binding compounds [ 92 , 93 ].…”