Formation of nucleosomes does not suppress interaction of a DNA fragment with an alkylating derivative of a pyrimidine oligonucleotide

Oligonucleotide derivatives capable of binding to specific nucleic acids are considered as potential therapeutic agents, exerting their action at the level of genome functioning (Hélène, 1991; Knorre et al., 1993). A straightforward approach to targeting DNA is based on using oligonucleotides capable of binding to oligopurine-oligopyrimidine sequences by formation of triple-strand structures. We report results of experiments on sequence-specific chemical modification of a 490-bp fragment of pfosCAT plasmid, containing the promoter segment of the c-fos gene using 4-(N-2-chloroethyl-N-methylamino)-benzylphosphamide derivatives of a homopyrimidine 14-mer oligonucleotide. It was shown that in both the free DNA and the DNA involved in nucleosome structure, reaction occurred with similar efficiency at the target guanosine residue G404.     

Refined high-field NMR solution structure of a binary-addressed pyrene/perfluoro-azide complementary DNA oligonucleotide system shows extensive distortion in the central nick region

The structural analysis of the photoactivated binary system of complementary-addressing nucleic acid sequences (1:2:3) by high-resolution NMR spectroscopy and restrained molecular dynamics is reported. The binary system comprised a 12 base-pair target DNA sequence, pdGTATCAGTTTCT (1), and two hexanucleotides, (dAGAAACp-L-Az (2) and Pyr-pdTGATAC (3)), complementary to neighbouring sites in the target DNA. Oligonucleotide (2) is conjugated with a p-azidotetrafludrobenzyl group (Az) via a linker group (L), and the other oligonucleotide (3) is equipped with the photosensitizing pyrenyl-1-methylamino group (Pyr). We now extend the structural analysis of 1:2:3, which was previously based on qualitative 2D 1H-NMR data and thermodynamic analysis of complex formation from UV-visible thermal denaturation experiments. In the current work structural refinement was performed by separate molecular dynamics runs for six different starting structures based on 318 proton-proton distance-range constraints, evaluated from the 1H-NOESY spectrum (tau(mix) = 200 ms, 600 MHz) using complete relaxation matrix analysis (NMR/TRIAD/MARDIGRAS). Additional Watson-Crick hydrogen bond restraints were included in the calculations based on the detected signals from the exchangeable protons, using REFOPT(NY) methods. The final averaged structure obtained from the six refined co-ordinate sets showed a considerable degree of axis bend (62.5 degrees) with the bending point in the middle of the duplex in the region of the backbone nick between the two short oligonucleotides. The complex behaves dynamically as the equivalent of two short B-DNA-like duplexes displaying a hinge-like flexing at their junction. In all final structures the Pyr function location was very restricted, the aromatic group lying in the duplex minor groove near residues 4T, 5C and 2T. In contrast, the location of the perfluoroazido group was different in all the final structures, indicating the high flexibility of this group in the duplex. The only feature common to all six final azido group orientations was the outside location on the side of the major groove. The distance between the Pyr and Az groups varied from 11 A to 24 A for the six final structures (17 A, final average structure). The dynamics of duplex denaturation for 1:2:3 was probed by monitoring the temperature-induced NMR line broadening of the imino protons in a 1D variable temperature NMR experiment. The melting of 1:2:3 starts both from the ends and from the middle part of the duplex at the backbone break between the two short oligonucleotides reflecting the destabilisation of the pyrene-arylazido nick region in the duplex.     

Endogenous oligonucleotides of human milk and their possible biological function.

Oligonucleotides (ON) 4 to 60 nucleotides in length were isolated by ion-exchange chromatography on a column with Fractogel TSK DEAE-650 (M) from human milk which was hydrolyzed with proteinase K. ON from 60 to 16 nucleotides were degraded by RNase A but were resistant to DNase I, and, thus, they were ribooligonucleotides. In the presence of [gamma-32P]ATP, ON and heparin inhibited the phosphorylation of 38- and 20-kD milk proteins and failed to affect the phosphorylation of a 76-kD protein. Human milk is believed to contain polyanion-dependent and polyanion-independent protein kinases. The influence of the ON on the activity of the cytotoxic fraction of human milk alpha-lactalbumin towards human mammary gland carcinoma MCF-7 cells was studied. The ON inhibited the cytostatic and cytotoxic effects of alpha-lactalbumin. Synthetic oligonucleotides and heparin had similar effects. The endogenous ON are suggested to be involved in the regulation of cytotoxic activity of human milk.     

The Rossmann fold of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a nuclear docking site for antisense oligonucleotides containing a TAAAT motif

The subcellular localisation of oligodeoxynucleotides (ODN) is a major limitation for their use against nuclear targets. In this study we demonstrate that an antisense ODN directed against cytosolic phospholipase A(2) (cPLA2) mRNA is efficiently taken up and accumulates in the nuclei of endothelial cells (HUVEC), human monocytes and HeLa cells. Gel shift experiments and incubation of cells with oligonucleotide derivatives show that the anti-cPLA2 oligo binds a 37 kDa protein in nuclear extracts. The TAAAT sequence was identified as the major binding motif for the nuclear protein in competition experiments with mutated ODNs. Modification of the AAA triplet resulted in an ODN which failed to localise in the nucleus. Moreover, inserting a TAAAT motif into an ODN localising in the cytosol did not modify its localisation. The 37 kDa protein was purified and identified after peptide sequencing as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). It was shown by confocal microscopy that GAPDH co-localises with anti-cPLA2 ODN in the nucleus and commercial GAPDH effectively binds the oligo. Competition experiments with increasing concentration of NAD(+) co-factor indicate that the GAPDH Rossmann fold is a docking site for antisense oligonucleotides containing a TAAAT motif.     

Release of nucleic acids by eukaryotic cells in tissue culture

Extracellular nucleic acids in cultures of A431 and HeLa cells were investigated. The data obtained demonstrate the presence of high weight DNA and RNA in the extracellular medium. Temporal changes of extracellular nucleic acids levels in growth medium were investigated.     

Downregulation of PGY1/MDR1 mRNA level in human KB cells by antisense oligonucleotide conjugates. RNA accessibility in vitro and intracellular antisense activity

Inhibition of PGY1/MDR1 (multidrug resistance gene 1) mRNA expression in multidrug resistant KB-8-5 cells by 5'-bis-pyrenyl-3'-aminohexyl oligodeoxyribonucleotide conjugates targeted to four sites of this mRNA has been investigated. Three of the tested oligonucleotide conjugates specifically inhibited the expression of PGY1/MDR1 mRNA as monitored by the RT-PCR assay. The oligonucleotide conjugate targeted to the region (+178; +194) of the PGY1/MDR1 mRNA decreased level of this mRNA to 10% compared to the control. Nuclease-resistant analogs of oligonucleotide, complementary to this MDR1 mRNA region therefore, might be considered as a prototype compounds for development of gene-targeted therapeutic agents for overcoming the MDR phenotype caused by the overexpression of the PGY1/MDR1 gene.     

Sensitized site-specific photomodification of ssDNA by binary systems of oligonucleotide conjugates: VI. Effect of substituents in the sensitizer anthracene residue

Photomodification of ssDNA by binary systems of oligonucleotide conjugates complementary to the adjacent sequences of the target DNA was studied. One of the conjugates comprised a substituted anthracene as a sensitizer; the other,p-azidotetrafluorobenzaldehyde 3-aminopropionylhydrazone as a photoreagent. The sensitized photomodification is initiated by the 365–580-nm light through an efficient energy transfer from the photoexcitated sensitizer onto the photoreagent in a complementary complex of the binary system with the DNA target where the sensitizer and the photoreagent are sterically converged. Influence of substituents in the anthracene residue on the efficiency of the DNA sensitized photomodification was considered. The oligonucleotide conjugate of anthracene-9-al 3-aminopropionylhydrazone allows highly specific initiation of the sensitized photomodification upon irradiation with visible light at >460 nm in conditions generating no photoreaction in the sensitizer’s absence. For Part V, see [1]; prefix “d” in designations of oligonucleotides is omitted.