ABSTRACT
Novel amino linker and spacer phosphoramidites were synthesized from methoxyoxalamido (MOX) percursors possessing a secondary hydroxyl, which when phosphitylated endowed stability to the corresponding phosphoramidites. The synthetic strategy is robust, and the chemistry is reactive towards a variety of primary aliphatic diamines and amino alcohols to produce distinctly unique phosphoramidites. The selection of building blocks determines the length and physico-chemical properties of the phosphoramidite tethering arms, and the synthesis can be specifically tailored to suit individual requirement.
Subject(s)
DNA, Intergenic/chemical synthesis , Organophosphorus Compounds , Amines , Drug Stability , Indicators and Reagents , Magnetic Resonance Spectroscopy , Molecular StructureABSTRACT
A number of novel biotin phosphoramidites, possessing exceptionally long and uncharged tethering arms, were synthesized from methoxyoxalamido (MOX) and succinimido (SUC) precursors. Included among these monomers is a uridine derivative with the biotin moiety attached through the 2'-position. Some of these phosphoramidites were used to make 5'-biotinylated primers, which were applied in direct sequencing of genomic DNA and capture of Sanger fragment pools.
Subject(s)
Amides/chemistry , Biotin , Oligodeoxyribonucleotides/chemical synthesis , Phosphoric Acids/chemistry , Amides/chemical synthesis , Base Sequence , Biotinylation , DNA/chemistry , Indicators and Reagents , Molecular Structure , Oligodeoxyribonucleotides/chemistry , Phosphoric Acids/chemical synthesis , Structure-Activity RelationshipABSTRACT
The CD4 receptor of T-helper cells is an essential participant in immune response formation and HIV infection. We report here that the extracellular domains of CD4 receptor can catalyze the phosphotransferase (kinase) reaction. Incubation of rsCD4 in solution with [gamma-32P]ATP results in the Ca2+-dependent autophosphorylation of the protein presumably at a His residue because the reaction is prevented by the diethylpyrocarbonate treatment. The rsCD4 phosphorylates milk casein or human plasma proteins as a Ser/Thr protein kinase.
Subject(s)
CD4 Antigens/chemistry , Protein Kinases/chemistry , Adenosine Triphosphate/metabolism , Animals , Binding Sites , CD4 Antigens/metabolism , Humans , Phosphorylation , Protein Kinases/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sequence Homology, Amino AcidABSTRACT
T7 RNA polymerase is shown to recognize the SP6 promoter including 17 base pairs before the transcription start site and produce the 5'-end TBEV RNA. The yield of TBEV RNA synthesized by heterologous T7 RNA polymerase from cDNA construction with SP6 promoter is higher than the RNA production by homologous SP6 RNA polymerase. The addition of 1 pmol template DNA with SP6 17 bp promoter in transcription mixture for SP6 or T7 RNA polymerases resulted in a 1-5 X 10(-2) pmol RNA production.
Subject(s)
DNA-Directed RNA Polymerases/metabolism , Encephalitis Viruses, Tick-Borne/genetics , Promoter Regions, Genetic/genetics , RNA, Viral/biosynthesis , Salmonella Phages/genetics , Transcription, Genetic , Bacteriophage T7/enzymology , Base Sequence , DNA, Complementary/metabolism , DNA, Viral/metabolism , Encephalitis Viruses, Tick-Borne/pathogenicity , Molecular Sequence Data , RNA, Viral/genetics , Salmonella Phages/enzymology , Salmonella typhimurium/virology , Viral ProteinsABSTRACT
The interaction of reactive derivatives of oligonucleotides bearing a 4-[(N-2-chloroethyl-N-methyl)amino]benzylamin residue at the 5'-terminal phosphate with serum blood proteins has been investigated. It was found that the compounds react with serum albumin and immunoglobulins M and G, the reactivity increasing in the order: albumin < IgG < IgM. The reactions with immunoglobulins were inhibited in the presence of different oligonucleotides, DNA and heparin, suggesting the oligonucleotide binding to some cationic region of the proteins. Myoglobin inhibited the interaction of oligonucleotide derivatives with myoglobin-specific monoclonal antibodies which indicates that the derivatives interact with the proteins within or near the antigen binding site.
Subject(s)
Blood Proteins/metabolism , Oligonucleotides/metabolism , Antibodies, Monoclonal/metabolism , Base Sequence , Binding Sites, Antibody , Binding, Competitive , DNA/pharmacology , Heparin/pharmacology , Humans , Immunoglobulin G/metabolism , Immunoglobulin M/metabolism , Molecular Sequence Data , Myoglobin/immunology , Myoglobin/pharmacology , Oligonucleotides/pharmacology , Serum Albumin/metabolismABSTRACT
Oligodeoxyribonucleotides (22-mers) were delivered through the skin of C3H mice in the region of a mammary gland tumor by means of iontophoresis. It was shown that the oligonucleotides enter the tumor, cross it, and reach all mouse organs. Electrophoretic analysis of the oligonucleotide extracted from tumor showed that the compounds were delivered in the tissue in the intact state.
Subject(s)
Mammary Neoplasms, Experimental/drug therapy , Oligonucleotides, Antisense/administration & dosage , Administration, Topical , Animals , Base Sequence , Iontophoresis , Mice , Mice, Inbred C3H , Molecular Sequence DataABSTRACT
Benzylamide 5'-32P-oligonucleotide derivatives were shown to penetrate into mice organism when administered by various routes; intranasally, per os, intravaginally and per rectum. In all cases, the compounds are rapidly accumulated in blood and guts. Analysis of the radioactive material from blood and pancreas revealed intact oligonucleotides. Although concentrations of oligonucleotides in tissues differ considerably by the various methods of administration, the efficiency of delivery is sufficient to consider all the routes as being of therapeutic value. Dose effect on the efficiency of oligonucleotide penetration into mice suggests the transport to be a saturable process. Application of an oligonucleotide lotion on mice ear helices results in reproducible accumulation of radioactivity in the animal tissues. Effectiveness of oligonucleotide delivery into mouse through skin can be improved by using electrophoretic procedure.
Subject(s)
Oligodeoxyribonucleotides/pharmacokinetics , Skin Absorption , Skin/metabolism , Animals , Base Sequence , Drug Administration Routes , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Mucous Membrane/metabolism , Oligodeoxyribonucleotides/administration & dosage , Phosphorus Radioisotopes , Tissue DistributionABSTRACT
Distribution and stability of benzyl-[5'-32P] phosphoramides of oligonucleotides and their phosphorothioate analogs were investigated. Oligonucleotide derivatives are distributed among all murine organs, the maximal concentration being observed in the liver and kidney, and minimal in the brain. Intravenous and intraperitoneal injections resulted in a faster distribution of oligonucleotides among the tissues than subcutaneous injections. Hydrolysis rate varied from tissue to tissue, and in 30 min after injection 5 to 50% of intact oligonucleotides were found. Hydrolysis did not depend on the injected dose in the range of 0.15-150 nmol per mouse. The 3'-terminal cholesterol groups protected the oligonucleotides considerably. In the blood stream and pancreas phosphorothioate oligonucleotides showed much higher stability than their phosphodiester counterparts. A protein interacting specifically with the oligonucleotides was discovered in the liver, kidney and pancreas.
Subject(s)
Oligonucleotides/pharmacokinetics , Animals , Base Sequence , Drug Stability , Kidney/metabolism , Liver/metabolism , Mice , Molecular Sequence Data , Oligonucleotides/chemistry , Organophosphorus Compounds/chemistry , Pancreas/metabolism , Tissue DistributionABSTRACT
Interactions of oligonucleotide derivatives with mammalian cells and cellular biopolymers have been investigated. The derivatives were oligonucleotides bearing an alkylating 2-chloroethylamino group at the 3'-end and a cholesterol residue at the 5'-terminal phosphate. These compounds are readily taken up by cells and react with cellular DNA, RNA and some proteins which may play a role in delivery of the compounds into cells.
Subject(s)
Biopolymers , Cholesterol/metabolism , Oligonucleotides/metabolism , Alkylation , Ascites/metabolism , Autoradiography , Cell Line , DNA/metabolism , Electrophoresis, Polyacrylamide Gel , Proteins/metabolism , RNA/metabolism , Tumor Cells, CulturedABSTRACT
A reaction of native Drosophila proteins with an alkylating oligonucleotide derivative bearing 4-[(N-2-chlorethyl-N-methyl)amino]benzylamine at the 5' terminal phosphate has been investigated. It was found, that the reagent alkylates a few proteins (90, 50, 44, 39, 32 kDa). The modification was organ specific. The labeled 39 kDa protein is present in the ovaries only, while the modified 32 kDa protein is found only in the bulbus.
