ABSTRACT
Uracyl and adenine containing oligocarboxamide mimetics of nucleic acids based on morpholine nucleosides (MorGly) are synthesized using peptide chemistry methods. Conditions for an analysis of homogeneity of protonated at physiological pH oligomers using a capillary electrophoresis are proposed. Studies of thermostability of complementary complexes formed by MorGly oligomers revealed that melting temperature dramatically depends on heterocyclic base composition (uracyl or adenine). Cooperative interactions realized at junctions in tandem complexes give more contribution to the thermostability in the case of complexes formed by modified oligomers than native oligodeoxyriboadenilates. Adenine containing MorGly oligomers form more stable complexes with poly(U) than native oligodeoxyriboadenilate of the same length. Complexes formed by modified oligomers with polyribonucleotides are more stable in compare with polydeoxyribonucleotide.
Subject(s)
Morpholinos , Nucleic Acids , Oligodeoxyribonucleotides , Adenine/chemistry , Morpholinos/chemical synthesis , Morpholinos/chemistry , Morpholinos/isolation & purification , Nucleic Acid Conformation , Nucleic Acids/chemical synthesis , Nucleic Acids/chemistry , Oligodeoxyribonucleotides/chemical synthesis , Oligodeoxyribonucleotides/chemistry , Oligodeoxyribonucleotides/isolation & purification , Uracil/chemistryABSTRACT
A simple and effective method for the synthesis of 2'-aminomethylmorpholino-4'-carboxymethyl nucleoside analogues and Boc-modified derivatives as synthons for peptide synthesis was developed.
Subject(s)
Morpholines/chemistry , Nucleotides/chemical synthesis , Oligonucleotides/chemical synthesis , Molecular StructureABSTRACT
To define of possible mechanism of monoclonal antibody B2 interaction with haptens and peptide-mimotope of benzo[a]pyrene Fab-fragment structure was modeled. Antibody active centre structure was defined using docking with a number of different polyclic aromatic hydrocarbons. It was shown that active center of monoclonal antibody B2 composed of two binding pockets. Correlation was revealed between experimental and calculated data on relative free energies of monoclonal antibody B2 binding with different ligands. We found that synthetic peptide-mimotope of benzo[a]pyrene weakly competeswith benzo[a]pyrene conjugate in monoclonal antibody B2 binding. Immunization of mice with peptide-mimotope conjugate did not revealed antibodies to benzo[a]pyrene. To define structural features of peptide-mimotope of benzo[a]pyrene preliminary model of peptide-mimotope in the frame of pIII protein was calculated. It was shown that tryptophan located inside of peptide can be exhibited on a surface and accessible to antibody. Obtained modeling results could be applied for following optimizations ofbenzo[a]pyrene peptide-mimotope and active center of monoclonal antibody B2.
Subject(s)
Antibodies, Monoclonal/chemistry , Benzo(a)pyrene/chemistry , Haptens/chemistry , Immunoglobulin Fab Fragments/chemistry , Models, Molecular , Peptides/chemistry , Animals , Antibodies, Monoclonal/immunology , Binding Sites, Antibody/immunology , Haptens/immunology , Immunoglobulin Fab Fragments/immunology , Mice , Mice, Inbred BALB C , Peptides/immunologySubject(s)
Genome, Viral/genetics , Histidine/analogs & derivatives , Influenza A Virus, H1N1 Subtype/genetics , Influenza A Virus, H1N1 Subtype/physiology , RNA, Viral/metabolism , Ribonucleases/metabolism , Virus Inactivation , Azabicyclo Compounds/chemistry , Azabicyclo Compounds/metabolism , Histidine/chemistry , Histidine/metabolism , Influenza A Virus, H1N1 Subtype/metabolism , Influenza A Virus, H1N1 Subtype/ultrastructure , Microscopy, Electron , Molecular Weight , Ribonucleases/chemistry , Virion/ultrastructureABSTRACT
The composition and kinetics of accumulation of extracellular DNA in cultures of primary human endotheliocytes, cervical adenocarcinoma, and mycoplasma-infected cervical adenocarcinoma cells were studied. The content of DNA bound to cell surface did not change during culturing. The concentration of extracellular DNA in culture medium increased during the lag phase and at the beginning of the exponential growth phase, which probably attests to active secretion of DNA by cells. Spontaneous extracellular DNA synthesis was observed only in cell culture infected with mycoplasma.
Subject(s)
DNA/analysis , Mycoplasma/isolation & purification , Cell Line, Transformed , Electrophoresis, Agar Gel , HeLa Cells , HumansABSTRACT
The dependence of hydrolytic activity of artificial ribonucleases toward an HIV-I RNA fragment, a 21-mer oligonucleotide, and tRNA Asp on the structure of the RNase mimetic was analyzed. The quantitative structure-activity relationship (QSAR task) was determined by the method of simplex representation of the molecular structure where the amounts of four-atom fragments (simplexes) of fixed structure, symmetry, and chirality served as descriptors. Not only the types of atoms participating in simplexes but also their physicochemical properties (e.g., partial charges, lipophilicities, etc.) were taken into account. This allowed the estimation of the relative role of various factors affecting the interaction of molecules under study with the corresponding biological target. The 2D QSAR models obtained by the method of projection to latent structures have quite satisfactory statistical indices (R2 = 0.82-0.96; Q2 = 0.73-0.89), which help predict the activities of new compounds. The electrostatic properties of ribonuclease atoms were shown to contribute significantly to the manifestation of the hydrolytic activity of ribonucleases in the case of the 21-mer oligonucleotide and tRNA. In addition, the structural fragments that most greatly contribute to the alteration of the hydrolytic activity of RNases were identified. The models obtained were used for the virtual screening and molecular design of new highly efficient RNase mimetics.
Subject(s)
Quantitative Structure-Activity Relationship , Ribonucleases/chemistry , Drug Design , HIV-1/genetics , Hydrolysis , Molecular Mimicry , Oligonucleotides/chemistry , RNA, Transfer, Asp/chemistry , RNA, Viral/chemistry , Substrate SpecificityABSTRACT
A simple method was developed for end-point fluorescence detection of the 735G --> A mutation of the 5'-splice donor site of intron 14 of the dihidropyrimidine dehydrogenase gene (DPYD). The method was based on allele-specific PCR with duplex Scorpion primers. The genotyping results obtained by the fluorescent endpoint PCR technique completely coincided with the results obtained by allele-specific PCR with amplicon detection in agarose gel. Genotyping was performed in 291 DNA samples from residents of Novosibirsk region (Russia), and two heterozygotes (0.69%) were detected.
Subject(s)
Alleles , DNA Primers/genetics , Dihydrouracil Dehydrogenase (NADP)/genetics , Point Mutation , RNA Splice Sites/genetics , DNA Primers/chemistry , Female , Fluorescent Dyes/chemistry , Genetic Carrier Screening/methods , Heterozygote , Humans , Male , Polymerase Chain Reaction , SiberiaABSTRACT
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
The effect of the total positive charge in the RNA-binding domain of chemical ribonucleases that are conjugates of bisquaternary salts of diazabicyclo[2.2.2]octane and imidazol on the cleavage of an HIV-1 RNA fragment was studied. An increase in the positive charge from +2 to +4 was shown to result in a significant growth in the ribonuclease activity. Possible mechanisms of the interactions between structural moieties of chemical ribonucleases and RNA that enable an effective catalysis of the cleavage of phosphodiester bonds are discussed.
Subject(s)
Imidazoles/chemistry , Piperazines/chemistry , RNA, Viral/chemistry , Ribonucleases/chemistry , Catalysis , HIV-1/genetics , Hydrophobic and Hydrophilic InteractionsABSTRACT
A number of monomers for the standard phosphoamidite oligodeoxynucleotide synthesis that carry reactive methoxyoxalamide groups attached to the thymidine, 2'-deoxycytidine, and 2'-deoxyadenosine heterocyclic bases were prepared. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 3; see also http://www.maik.ru.
Subject(s)
Amides/chemistry , Deoxyribonucleosides/chemical synthesis , Oligodeoxyribonucleotides/chemical synthesis , Oxamic Acid/chemistry , Deoxyribonucleosides/chemistry , Heterocyclic Compounds/chemistry , Oligodeoxyribonucleotides/chemistryABSTRACT
A new photoreactive oligonucleotide derivative was synthesized with a perfluoroarylazido group attached to the 2'-position of the ribose fragment of the 5'-terminal nucleotide. Using this conjugate, photoreactive DNA duplexes were produced which contained single-stranded regions of different length, single-stranded breaks (nicks), and also ds duplex with a photoreactive group inside one of the chains. These structures imitate DNA intermediates generated at different stages of DNA replication and repair. The interaction of replication protein A (RPA) with the resulting DNA structures was studied using photoaffinity modification and gel retardation assay. Independently of the DNA structure, only the large subunit of RPA (p70) was crosslinked to photoreactive DNAs, and the intensity of its labeling increased with decrease in the size of the single-stranded region and was maximal in the case of the nick-containing DNA structure. By gel retardation, the most effective binding of RPA to this structure was shown, whereas the complexing of RPA with DNA containing the unmodified nick and also with the full duplex containing the photoreactive group inside the chain was significantly less effective. The data suggest that RPA should be sensitive to such damages in the double-stranded DNA structure.
Subject(s)
Azides/chemistry , Benzoates/chemistry , DNA, Single-Stranded/chemistry , DNA-Binding Proteins/chemistry , Nucleotides/chemistry , Oligonucleotides/chemistry , Oligonucleotides/chemical synthesis , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Base Sequence , Binding Sites , DNA Damage , DNA Replication , DNA-Binding Proteins/metabolism , Escherichia coli , Molecular Sequence Data , Oligonucleotides/metabolism , Protein Binding , Replication Protein A , Substrate SpecificityABSTRACT
Kinetic parameters of cleavage of CpA and UpA sequences in an oligoribonucleotide under the action of artificial ribonuclease ABL3C1 were measured. The compounds were built of RNA-binding domain B, catalytic fragment C, linker L3 comprising 3 methylene groups, and aliphatic fragment A. The rate of cleavage of phosphodiester bonds in CpA sequence within decaribonucleotide UUCAUGUAAA was shown to be 3.4 +/- 0.2 times higher than in UpA sequence. The rate of cleavage of phosphodiester bonds were found to depend on substrate length: a thousandfold increase in cleavage rate constant was observed for CpA sequence in decaribonucleotide as compared with diribonucleotide monophosphate CpA. A slight decrease in the cleavage rates was observed for the reactions proceeding in different buffers at pH 7.0: imidazole > HEPES > phosphate > cacodylate. At the same time, the ratio of cleavage rates for CpA and UpA sequences remained constant.
Subject(s)
Aza Compounds/chemistry , Bridged Bicyclo Compounds, Heterocyclic/chemistry , Imidazoles/chemistry , Oligoribonucleotides/chemistry , Ribonucleases/chemistry , Binding Sites , Catalysis , Dinucleoside Phosphates/chemistry , Hydrolysis , Kinetics , Molecular Mimicry , RNA/metabolism , Ribonuclease, Pancreatic/chemistry , Structure-Activity RelationshipABSTRACT
A method has been suggested for the synthesis of conjugates of oligodeoxyribonucleotides with chemical constructs mimicking ribonuclease A active center for directed fragmentation of RNA. The method is based on the sequential addition of linker group, 9-(methylamino)anthracene, to 5' or 3' terminal phosphate of oligonucleotide and then imidazole-containing construct by cycloaddition reaction. The conjugates of oligonucleotides complementary to regions 44-61 (2B-R) and 60-76 (1C-R) of yeast phenylalanine tRNA demonstrated ability to cleave tRNA(Phe) under physiological conditions preferably at the sole phosphodiester bond (C63-A64 for 2B-R and C56-G57 for 1C-R, respectively). The half-time of tRNA(Phe) hydrolysis in the presence of 2B-R conjugate was 30 min at 2B-R concentration of 10 microM and several minutes at conjugate concentration of 50 microM.
Subject(s)
Biochemistry/methods , Imidazoles/chemistry , Oligonucleotides, Antisense/chemistry , RNA/chemistry , Base Sequence , Hydrolysis , Kinetics , Molecular Sequence Data , RNA, Transfer, Phe/chemistry , Ribonuclease, Pancreatic/chemistryABSTRACT
Artificial ribonucleases of the ABLkCm series were synthesized. They consist of a lipophilic alkyl radical (Et, n-C14H29, or C15H31) A, an "RNA-binding domain" B (bisquaternary salt of 1,4-diazabicyclo[2.2.2]octane), a "catalytic domain" Cm [histamine (C1) or histidine (C3) residue], and a "linker" Lk that joins the "domains" B and Cm [here, k is the number of methylene units (one or three) in the linker]. The effect of the "domain structure" on the catalytic properties of the chemical ribonucleases was analyzed using seven compounds of this series (ABL1C1, ABL3C1, ABL3C3, AC1, AB, BL2, and BL3C3). The catalytic activity of the compounds was assessed in the reaction of hydrolysis of the in vitro transcripts of human tRNA(Lys) and yeast tRNA(Asp) under physiological conditions. It was shown that only chemical ribonucleases that involve all the fragments of the ABLkCm construct can hydrolyze the substrate tRNA at a high rate (90% of tRNA is hydrolyzed for 10 h at 37 degrees C). The activity of the compounds is largely determined by the presence of a long lipophilic radical linked to 1,4-diazabicyclo[2.2.2]octane and a long linker, which joins the RNA-hydrolyzing and RNA-binding fragments. The results indicate an important role of hydrophobic interactions in the acceleration of the RNA hydrolysis reaction. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2002, vol. 28, no. 4; see also http://www.maik.ru.
Subject(s)
Piperazines/chemistry , Ribonucleases/chemical synthesis , Catalysis , Humans , Hydrolysis , Kinetics , Nucleic Acid Conformation , RNA, Transfer, Asp/chemistry , RNA, Transfer, Lys/chemistry , Yeasts/chemistryABSTRACT
A procedure was proposed allowing one to synthesize RNA mimics on the basis of conjugates of diazabicyclo[2.2.2]octane with imidazole bearing a varying number of positive charges (nDm series, where n is the number of positive charges at neutral pH, m is the code of an imidazole-containing fragment of the catalytic domain: 1, histamine; 2, histidine methyl ester). The hydrolytic activity of six compounds of this series was studied under physiological conditions using in vitro transcript of human mitochondrial tRNA(Lys) as a substrate. It was shown that the rate of RNA hydrolysis with nDm conjugates rises with an increase in the number of positive charges: an approximately 30-fold acceleration of hydrolysis was observed with an increase in the total charge of the construct from +2 to +4.
Subject(s)
Bridged Bicyclo Compounds, Heterocyclic/chemical synthesis , Imidazoles/chemical synthesis , Ribonucleases/chemistry , Bridged Bicyclo Compounds, Heterocyclic/chemistry , Catalysis , Cations, Monovalent/chemistry , Drug Design , Humans , Hydrolysis , Imidazoles/chemistry , Magnesium/chemistry , Mitochondria/chemistry , Molecular Mimicry , Point Mutation , RNA, Transfer, Lys/chemistry , RNA, Transfer, Lys/genetics , Structure-Activity RelationshipABSTRACT
An oligonucleotide conjugate bearing a chemical construct mimicking the catalytic center of ribonuclease A has been designed and studied. The conjugate efficiently cleaves yeast tRNAPhe at a single site adjacent to the target complementary sequence.
Subject(s)
Molecular Mimicry , Oligonucleotides, Antisense/chemistry , RNA, Complementary/chemistry , RNA, Transfer, Phe/chemistry , Ribonucleases/chemistry , Anthracenes/chemistry , Base Sequence , Catalysis/drug effects , Hydrolysis/drug effects , Imidazoles/chemistry , Molecular Sequence Data , Oligonucleotides, Antisense/chemical synthesis , RNA, Fungal/chemistry , RNA, Transfer, Phe/antagonists & inhibitors , Saccharomyces cerevisiae , Thionucleotides/chemistrySubject(s)
RNA/metabolism , Ribonucleases/chemistry , Base Sequence , Electrophoresis, Polyacrylamide Gel , Genetic Techniques , Hydrolysis , Imidazoles/pharmacology , Models, Chemical , Molecular Sequence Data , Nucleic Acid Conformation , Nucleic Acid Hybridization , Phosphorylation , Plasmids/metabolism , RNA/chemistry , RNA, Transfer, Asp/chemistry , Ribonuclease, Pancreatic/chemistry , Ribonuclease, Pancreatic/pharmacology , Ribonucleases/pharmacology , Saccharomyces cerevisiae/genetics , SpectrophotometryABSTRACT
On the basis of imidazole and bisquaternary salts of 1,4-diazabicyclo[2.2.2]octane, a number of highly effective catalysts of the nDm series (here, n is the number of positive charges at neutral pH values and m is the digital code of the catalytically active fragment: 1, histamine, and 2, histidine methyl ester) were synthesized for the cleavage of the phosphodiester bonds in ribonucleic acids. A general method for the synthesis of chemical ribonucleases was suggested, which helps vary both the number of positive charges in their RNA-binding domain and the catalytic center. By the example of hydrolysis under physiological conditions of the in vitro transcript of tRNA(Lys) from human mitochondria, it was shown that the RNA cleavage rate with the nDm conjugates increases approximately 30-fold along with the increase in the number of positive charges from two to four.