ABSTRACT
New zwitter-ionic oligonucleotide derivatives containing 1,2,3,4-tetrahydroisoquinoline-7-sulfonyl phosphoramidate group are described. Automated synthesis of these compounds was carried out according to the ß-cyanoethyl phosphoramidite scheme via the Staudinger reaction between 2-trifluoroacetyl-1,2,3,4-tetrahydroisoquinoline-7-sulfonyl azide and phosphite triester within oligonucleotide grafted to polymer support. 1,2,3,4-Tetrahydroisoquinoline-7-sulfonyl phosphoramidate group (THIQ) was stable under the conditions of standard oligonucleotide synthesis, including the removal of protective groups and cleavage of the oligonucleotide from the polymer support by treatment with a mixture of concentrated aqueous solutions of ammonia and methylamine (1 : 1) at 55°C. Oligonucleotides modified by one to five THIQ groups in various positions were obtained. The zwitter-ionic character of the obtained derivatives was reflected in their varying mobility under conditions of denaturing PAGE. The thermal stability of the duplexes of oligodeoxynucleotides containing THIQ groups with complementary DNA and RNA only slightly differed from that of natural DNA:DNA and DNA:RNA duplexes. The results reported suggest that oligonucleotides modified with zwitter-ionic THIQ groups as antisense therapeutic agents.
Subject(s)
DNA , Oligonucleotides , Oligonucleotides/chemistry , DNA/chemistry , RNA/chemistry , PolymersABSTRACT
Myeloid dendritic cells (DCs) play an important role in the immune response; therefore, the search for compounds that can effectively activate DCs is a needful goal. This study was aimed to investigate the effect of synthetic CpG oligodeoxynucleotides (CpG-ODN) on the maturation and allostimulatory activity of myeloid DCs in comparison with other PAMP and DAMP molecules. For the research, we synthesized known CpG-ODN class C (SD-101 and D-SL03) containing thiophosphate internucleotide groups, and their original phosphate-modified analogues (SD-101M and D- SL03M) with mesylphosphoramide internucleotide groups (M = µ-modification). The effects of CpG-ODN and other activators were evaluated on DCs generated from blood monocytes in the presence of GM-CSF and IFN-α (IFN-DC) or IL-4 (IL4-DC). Evaluation of the intracellular TLR-9 expression showed that both types of DCs (IFN-DC and IL4-DC) contained on average 52 and 80 % of TLR-9-positive cells, respectively. The CpG-ODNs studied enhanced the allostimulatory activity of IFN-DCs, and the effect of µ-modified CpG-ODNs was higher than that of CpG-ODNs with thiophosphate groups. The stimulating effect of CpG-ODN at a dose of 1.0 µg/ml was comparable (for D-SL03, D-SL03M, SD-101) with or exceeded (for SD-101M) the effect of LPS at a dose of 10 µg/ml. At the same time, IFN-DCs were characterized by greater sensitivity to the action of CpG-ODNs than IL4-DCs. The enhancement of DC allostimulatory activity in the presence of CpG-ODNs was associated with the induction of final DC maturation, which was confirmed by a significant decrease in the number of CD14+DC, an increase in mature CD83+DC and a trend towards an increase in CD86+DC. Interestingly, the characteristic ability of LPS to enhance the expression of the co-stimulatory molecule OX40L on DCs was revealed only for the µ-analogue SD-101M. In addition, CpG-ODNs (SD-101 and SD-101M) had a stimulatory effect on IFN-γ production comparable to the action of LPS. The data obtained indicate a stimulating effect of CpG-ODN on the maturation and allostimulatory activity of human myeloid DCs, which is more pronounced for µ-modified analogs.
ABSTRACT
Here we describe a DNA analog in which the mesyl (methanesulfonyl) phosphoramidate group is substituted for the natural phosphodiester group at each internucleotidic position. The oligomers show significant advantages over the often-used DNA phosphorothioates in RNA-binding affinity, nuclease stability, and specificity of their antisense action, which involves activation of cellular RNase H enzyme for hybridization-directed RNA cleavage. Biological activity of the oligonucleotide analog was demonstrated with respect to pro-oncogenic miR-21. A 22-nt anti-miR-21 mesyl phosphoramidate oligodeoxynucleotide specifically decreased the miR-21 level in melanoma B16 cells, induced apoptosis, reduced proliferation, and impeded migration of tumor cells, showing superiority over isosequential phosphorothioate oligodeoxynucleotide in the specificity of its biological effect. Lower overall toxicity compared with phosphorothioate and more efficient activation of RNase H are the key advantages of mesyl phosphoramidate oligonucleotides, which may represent a promising group of antisense therapeutic agents.
Subject(s)
Amides/metabolism , Oligonucleotides, Antisense/metabolism , Oligonucleotides/metabolism , Phosphates/metabolism , Phosphoric Acids/metabolism , Animals , Apoptosis/genetics , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation/genetics , DNA/metabolism , Melanoma, Experimental , Mice , MicroRNAs/metabolism , RNA/metabolism , Ribonuclease H/metabolismABSTRACT
In the present study, we designed and synthesised new polycationic molecules based on two 1,4-diazabicyclo[2.2.2]octane (DABCO) moieties with hydrophobic groups connected by different linkers. The structure and the RNA-cleavage activity relationships of this novel series of artificial ribonucleases (aRNases) were investigated.
Subject(s)
Piperazines/chemistry , Piperazines/pharmacology , RNA/metabolism , Ribonucleases/chemistry , Piperazines/chemical synthesis , Polyamines/chemical synthesis , Polyamines/chemistry , Polyamines/pharmacology , Polyelectrolytes , Structure-Activity RelationshipABSTRACT
A number of tetracationic compounds capable of phosphodiester bond cleavage within a 21 -membered ribooligonucleotide were designed and synthesized. The artificial ribonucleases represent two residues of quaternized 1,4-diazabicyclo[2.2.2]octane bearing alkyl substituents of various lengths and connected with a rigid linker. The efficiency of cleavage of phosphodiester bonds in an RNA target depends on the linker structure and the length of alkyl substituent.
Subject(s)
Piperazines/chemistry , Polyamines/chemistry , RNA/chemistry , Ribonucleases/chemistry , Base Sequence , Catalysis , Hydrolysis , Magnetic Resonance Spectroscopy , Molecular Structure , Oligoribonucleotides/chemistry , Polyamines/chemical synthesis , Polyelectrolytes , Ribonucleases/chemical synthesisABSTRACT
Combinatorial libraries of small organic compounds capable of cleaving RNA were synthesized. The compounds contain benzene ring substituted with two residues of bis quaternary salt of diazabicyclo[2.2.2]octane (DABCO) bearing hydrophobic fragments of different length and structure, attached to DABCO at the bridge position. These compounds, lacking traditional functionalities involved in transesterification reaction, exhibit pronounced RNA cleavage activity. To identify the most active artificial ribonucleases, sublibraries and truncated libraries, containing compounds lacking one of substituents were synthesized. Analysis of ribonuclease activity of truncated libraries resulted in identification of the most active compounds, which are characterized by the presence of at least one long oligomethylene substituent.
Subject(s)
Combinatorial Chemistry Techniques/methods , Piperazines/chemistry , RNA/chemistry , Ribonucleases/chemistry , Alkanes/chemistry , Benzene Derivatives/chemical synthesis , Benzene Derivatives/chemistry , Catalysis , Oligoribonucleotides/chemistry , RNA/metabolism , Ribonucleases/metabolismABSTRACT
Cationic compounds containing benzene ring substituted with the bis-quaternary salt of diazabicyclo[2.2.2]octane (DABCO) bearing a polymethylene fragment at the bridge positions display ribonuclease activity. Efficacy of the catalysis is affected by geometry of the cationic structures and the size of the attached aliphatic fragment. The cleavage occurs primarily within CA sequences. The compounds do not possess tradition groups participating in the transesterification step of RNA cleavage reaction, therefore a speculative mechanism of cleavage could be inducing a conformational stress on the RNA sugar phosphate backbone providing fragility to phosphodiester bonds.