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Phosphodiester Backbone (phosphodiester + backbone)
Selected AbstractsToll-like receptor 9 binds single-stranded CpG-DNA in a sequence- and pH-dependent mannerEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 9 2004Mark Rutz Abstract Toll-like receptors (TLR) recognize bacterial and viral components, but direct interaction of receptor and ligand is unclear. Here, we demonstrate that TLR9 binds directly and sequence-specifically to single-stranded unmethylated CpG-DNA containing a phosphodiester backbone. TLR9-CpG-DNA interaction occurs at the acidic pH (6.5,5.0) found in endosomes and lysosomes. By sequence comparison we identified a potential CpG-DNA binding domain homologous to that described for methyl-CpG-DNA binding proteins. Amino acid substitutions in this region abrogated CpG-DNA binding and led to loss of NF-,B activation. Furthermore, chloroquine and quinacrine, therapeutic agents for autoimmune diseases like rheumatoid arthritis and systemic lupus erythematosus, directly blocked TLR9-CpG-DNA interaction but not TLR2-Pam3Cys binding. Our results demonstrate direct binding of TLR9 to CpG-DNA and suggest that the therapeutic activity of chloroquine and quinacrine in autoimmune diseases may be due to its activity as a TLR9 antagonist and inhibitor of endosomal acidification. [source] Shear-induced degradation of plasmid DNAJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 7 2002C. S. Lengsfeld Abstract The majority of gene therapy clinical trials use plasmid DNA that is susceptible to shear-induced degradation. Many processing steps in the extraction, purification, and preparation of plasmid-based therapeutics can impart significant shear stress that can fracture the phosphodiester backbone of polynucleotides, and reduce biological activity. Much of the mechanistic work on shear degradation of DNA was conducted over 30 years ago, and we rely heavily on this early work in an attempt to explain the empirical observations of more recent investigations concerning the aerosolization of plasmids. Unfortunately, the sporadic reports of shear degradation in the literature use different experimental systems, making it difficult to quantitatively compare results and reach definitive mechanistic conclusions. In this review, we describe the forces imparted to DNA during shear stress, and use published data to quantitatively evaluate their relative effects. In addition, we discuss the effects of molecular weight, strain rate, particle size, flexibility, ionic strength, gas,liquid interfaces, and turbulence on the fluid flow degradation of supercoiled plasmid DNA. Finally, we speculate on computational methods that might allow degradation rates in different experimental systems to be predicted. © 2002 Wiley-Liss Inc. and the American Pharmaceutical Association J Pharm Sci 91:1581,1589, 2002 [source] Head-to-Head Right-Handed Cross-Links of the Antitumor-Active Bis(,- N,N,-di- p -tolylformamidinato)dirhodium(II,II) Unit with the Dinucleotides d(GpA) and d(ApG)CHEMISTRY - A EUROPEAN JOURNAL, Issue 32 2008Helen Abstract Reactions of cis -[Rh2(DTolF)2(NCCH3)6](BF4)2 with the dinucleotides d(GpA) and d(ApG) proceed to form [Rh2(DTolF)2{d(GpA)}] and [Rh2(DTolF)2{d(ApG)}], respectively, with bridging purine bases spanning the Rh,Rh unit in the equatorial positions. Both dirhodium adducts exhibit head-to-head (HH) arrangement of the bases, as indicated by the presence of H8/H8 NOE cross-peaks in the 2D ROESY NMR spectra. The guanine bases bind to the dirhodium core at positions N7 and O6, a conclusion that is supported by the absence of N7 protonation at low pH,values and the notable increase in the acidity of the guanine N1H sites (pKa,7.4 in 4:1 CD3CN/D2O), inferred from the pH-dependence titrations of the guanine H8 proton resonances. In both dirhodium adducts, the adenine bases coordinate to the metal atoms through N6 and N7, which induces stabilization of the rare imino tautomer of the bases with a concomitant substantial decrease in the basicity of the N1H adenine sites (pKa,7.0,7.1 in 4:1 CD3CN/D2O), as compared to the imino form of free adenosine. The presence of the adenine bases in the rare imino form is further corroborated by the observation of DQF-COSY H2/N1H and ROE N1H/N6H cross-peaks in the 2D NMR spectra of [Rh2(DTolF)2{d(GpA)}] and [Rh2(DTolF)2{d(ApG)}] in CD3CN at ,38,°C. The 2D NMR spectroscopic data and the molecular modeling results suggest the presence of right-handed variants, HH1R, in solution for both adducts (HH1R refers to the relative base canting and the direction of propagation of the phosphodiester backbone with respect to the 5, base). Complete characterization of [Rh2(DTolF)2{d(GpA)}] and [Rh2(DTolF)2{d(ApG)}] by 2D,NMR spectroscopy and molecular modeling supports anti- orientation of the sugar residues for both adducts about the glycosyl bonds as well as N- and S-type conformations for the 5,- and 3,-deoxyribose residues, respectively. [source] Off-Target Effects Related to the Phosphorothioate Modification of Nucleic AcidsCHEMMEDCHEM, Issue 8 2010Johannes Winkler Dr. Abstract Phosphorothioate antisense oligonucleotides have been widely used in clinical studies for rational sequence-specific gene silencing. However, several sequence-unspecific off-target effects have been recently described for this compound class. In contrast to siRNA-mediated knockdown of the same gene, the bcl-2 -targeted oblimersen (Genasense, G3139) downregulates a number of proteins involved in apoptotic resistance and several glycolytic enzymes in 607B human melanoma cells. Regardless of their target, phosphorothioate-modified antisense and siRNA compounds, but not oligonucleotides with a phosphodiester backbone, resulted in a similar impact on the proteome. Unspecifically downregulated proteins include cancer markers involved in apoptotic resistance and endoplasmatic reticulum (ER) stress such as the 78,kDa glucose regulated protein (GRP,78), protein disulfide isomerase,A3 (PDIA3, GRP,58), calumenin, and galectin-1, as well as the glycolytic enzymes triose phosphate isomerase, glyceraldehyde phosphodehydrogenase, and phosphoglycerate mutase. The depletion of the glycolytic enzymes is reflected by a decrease in L -lactate production, indicating a partial reversal of the Warburg effect. Compared with other phosphorothioate oligonucleotides, oblimersen generally led to a more pronounced effect both in terms of the number of influenced proteins and the extent of downregulation, suggesting a synergistic effect of Bcl-2 downregulation. [source] Use of A-type CpG oligodeoxynucleotides as an adjuvant in allergen-specific immunotherapy in humans: a phase I/IIa clinical trialCLINICAL & EXPERIMENTAL ALLERGY, Issue 4 2009G. Senti Summary Background B-type CpG oligodeoxynucleotides (ODN) is currently used in clinical trials because of its prolonged half,life, which is due to its phosphorothioate backbone. A-type CpG ODN is a stronger inducer of IFN but has an unstable phosphodiester backbone that has so far prohibited its clinical use. However, upon association with virus-like particles (VLP) consisting of the bacteriophage Q, coat protein, A-type CpG ODN can be stabilized and can become an efficient adjuvant in mice. Therefore, the phase I/IIa study presented represents the first test of A-type CpGs in humans. Objective To test the safety, tolerability and clinical efficacy of QbG10 as an adjuvant for subcutaneous immunotherapy with a house dust mite (HDM) allergen extract in allergic patients. Methods A single centre, open-label phase I/IIa study evaluated the safety, tolerability and clinical efficacy of QbG10 as an adjuvant to immunotherapy with a subcutaneous HMD allergen extract in 20 patients suffering from HDM allergy. Twenty-one patients were enrolled between March and July 2005. Individual immunotherapy lasted 10 weeks. Clinical end-points included questionnaires, conjunctival provocation, skin prick tests and the measurement of allergen-specific IgG and IgE. Results QbG10 was well tolerated. Almost complete tolerance to the allergen was observed in conjunctival provocation testing after treatment with QbG10, and symptoms of rhinitis and allergic asthma were significantly reduced. Within 10 weeks of therapy, patients were nearly symptom-free and this amelioration lasted for at least 38 weeks post-treatment. Following injections of QbG10 and HDM allergen extract, allergen-specific IgG increased, while there was a transient increase in allergen-specific IgE titres. Skin reactivity to HDM was reduced. Conclusion The subcutaneous application of HDM allergen, together with A-type CpG ODN packaged into VLP, was safe. All patients achieved practically complete alleviation of allergy symptoms after 10 weeks of immunotherapy. This promising clinical outcome calls for larger placebo-controlled phase II studies. [source] The NMR structure of [Xd(C2)]4 investigated by molecular dynamics simulationsMAGNETIC RESONANCE IN CHEMISTRY, Issue 1 2003Thérèse E. Malliavin Abstract The i-motif tetrameric structure is built up from two parallel duplexes intercalated in a head-to-tail orientation, and held together by hemiprotonated cytosine pairs. Two topologies exist for the i-motif structure, one with outermost 3, extremities and the other with outermost 5, extremities, called the 3,E and 5,E topology, respectively. Since the comparison of sugar and phosphate group interactions between the two topologies is independent of the length of the intercalation motif, the relative stability of the 3,E and 5,E topologies therefore should not depend on this length. Nevertheless, it has been shown that the 3,E topology of the [d(C2)]4 is much more stable than the 5,E topology, and that the former is the only species observed in solution. In order to understand the reason for this atypical behavior, the NMR structure of the [Xd(C2)]4 was determined and analyzed by molecular dynamics simulations. In the NMR structure, the width of the narrow groove is slightly smaller than in previously determined i-motif structures, which supports the importance of phosphodiester backbone interactions in the structure stability. The simulations show that the stacking of cytosines, essential for the i-motif stability, is produced by a similar and non-negative twisting of the phosphodiester backbones. The twisting is induced by an interaction between the backbones; the [Xd(C2)]4 in 5,E topology, exhibiting very limited interaction between the phosphodiester backbones, is thus unstable. Copyright © 2002 John Wiley & Sons, Ltd. [source] | |||||||||||||