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Biomedical subjects

Z A Shabarova

Publications and source records attributed to Z A Shabarova.

At least 19 recordsLinked to original sources
PubMed2000

Targeting of single-stranded DNA and RNA containing adjacent pyrimidine and purine tracts by triple helix formation with circular and clamp oligonucleotides.

The aim of this work was to construct an anti-messenger targeted to the pim-1 oncogene transcript, based on circular or clamp oligodeoxyribonucleotides. The formation of bimolecular triplexes by clamp or circular oligonucleotides was investigated using single-stranded targets of both DNA (5'-CCCTCCTTTGAAGAA-3') and RNA type (5'-CCCUCCUUUGAAGAA-3'). The third, 'Hoogsteen' strand of the triplex was represented by G,T-rich sequences. The secondary structures of the complexes were determined by thermal denaturation, circular dichroism and gel mobility shift experiments and shown to depend on the nature of the target strand. With DNA as target, the sequence of a clamp (or circular) oligonucleotide that formed the triple helix was 3'-GGGAGGAAACTTCTTTT-TTGTTGTTT-TT-GGTGGG-5', where the first TT dinucleotide (in italics) is a linker and the second TT (bold) represents the bridge through which the 'Hoogsteen' strand switches from one strand of the Watson-Crick duplex to the other, once the duplex is formed by the corresponding portion of the anti-messenger (underlined). The portion of the 'Hoogsteen' sequence of the triplex between the two TT dinucleotides binds to the 3' extremity of the target strand and runs parallel to it. The portion situated at the 5' end of the oligonucleotide switches to the purine tract of the complementary strand of the duplex and is antiparallel to it. In contrast, with RNA as target, for a branched clamp oligonucleotide that formed a triple helix over its entire length (5'-TTCTTCAAAGGAGGG-3' 3'-GGGTGGTTT-T-GTTGTT-5') the portion of the 'Hoogsteen' sequence that bound to the 3' extremity of the target strand had to be antiparallel to it.

Circular Dichroism↗
PubMed1999

Oligonucleotide-peptide conjugates as potential antisense agents.

Oligonucleotide-peptide conjugates have several applications, including their potential use as improved antisense agents for interfering with the RNA function within cells. In order to provide robust and generally applicable conjugation chemistry, we developed a novel approach of fragment coupling of pre-synthesized peptides to the 2'-position of a selected nucleotide within an otherwise protected oligonucleotide chain attached to a solid support.

Adenosine↗
PubMed1999

Physico-chemical and biological properties of antisense phosphodiester oligonucleotides with various secondary structures.

The influence of the secondary structure of oligonucleotides having a natural phosphodiester backbone on their ability to interact with DNA and RNA targets and on their resistance to the nucleolytic digestion is investigated. Oligonucleotides having hairpin, looped and snail-like structure are found to be much more stable to nuclease degradation in different biological media and inside cells than the linear ones. The structured oligonucleotides can also hybridise with their DNA and RNA targets.

3T3 Cells↗
PubMed1999

A model of EcoRII restriction endonuclease action: the active complex is most likely formed by one protein subunit and one DNA recognition site.

To elucidate the mechanism of interaction of restriction endonuclease EcoRII with DNA, we studied by native gel electrophoresis the binding of this endonuclease to a set of synthetic DNA-duplexes containing the modified or canonical recognition sequence 5'-d(CCA/TGG)-3'. All binding substrate or substrate analogues tested could be divided into two major groups: (i) duplexes that, at the interaction with endonuclease EcoRII, form two types of stable complexes on native gel in the absence of Mg2+ cofactor; (ii) duplexes that form only one type of complex, observed both in the presence and absence of Mg2+. Unlike the latter, duplexes under the first group can be hydrolyzed by endonuclease. Data obtained suggest that the active complex is most likely formed by one protein subunit and one DNA recognition sequence. A model of EcoRII endonuclease action is presented.

Base Sequence↗
PubMed1999

Efficient synthesis of DNA dumbbells using template-induced chemical ligation in double-stranded polynucleotides closed by minihairpin fragments.

The chemical ligation of 17 50-54-membered nicked DNA dumbbells with different closing fragments, nick positions, and nucleotides facing the nick were investigated. T4, T5, GTA4C, GCGA2GC, and GCGA3GC sequences were chosen as the closing fragments. The nicks were placed in the center of the duplex stem or were adjacent to the closing fragments. N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide and cyanogen bromide were used as the condensing agents. We showed that the ligation efficiency is 10%-90% depending on the sequence of the closing fragments, nick position, and nucleotides facing the nick. Coupling yields of 80%-90% were observed when the nick was situated in the middle of the molecule between two T residues or was adjacent to GCGA2GC or GCGA3GC minihairpins. In the last case, the reacting 3'-phosphate and 5'-hydroxy groups were brought close together by only two base pair minihairpins. The coupling yields did not depend on the nature of the condensing agent. On the basis of the results obtained, we believe a rational design of nicked DNA dumbbells has been developed for efficient chemical synthesis of closed dumbbells.

Base Pairing↗
PubMed1998

Design and synthesis of double-stranded oligonucleotides containing reactive acylphosphate internucleotide groups.

DNA duplex and dumbbells containing chemically active acylphosphate internucleotide groups were synthesized. To obtain these compounds the chemical ligation method was used. The acylphosphate group was inserted into a DNA duplex and dumbbells as a result of template-directed condensation of 5'-phosphate and especially introduced 3'-carboxy groups of oligonucleotides. 1-Ethyl-3-(3'-dimethylaminopropyl)carbodiimide (EDC) was used as a condensing agent. Oligonucleotides containing a carboxy group were obtained by the interaction of their 3'-phosphate with glycine methyl ester under the action of EDC, followed by ester hydrolysis. The yields of acylphosphate-containing double-stranded oligonucleotides achieved 15-25% depending on the structure of their precursors. It was shown that these compounds are acylating agents and are efficiently cleaved in near-physiological conditions under the action of ethylenediamine or N-methylimidazole. These results indicate that double-stranded oligonucleotides carrying acylphosphate internucleotide groups could constitute new crosslinking reagents for affinity modification of DNA recognizing proteins.

Base Sequence↗
PubMed1998

Transition-metal complexes as inhibitors of proteins recognizing double-stranded fragments of nucleic acids.

A 30-membered DNA duplex containing the recognition site of restriction endonuclease SsoII and a 30-membered RNA--DNA hybrid duplex, a substrate of E. coli RNase H, were synthesized. The cleavage of the 30-membered fragments of nucleic acids catalyzed by endonucleases in the presence of Co-phthalocyanine complex [CoPc(COONa)8] containing eight carboxyl groups at the periphery of the ligand was studied. It was shown that the efficiency of enzyme catalysis decreases in the presence of the metal complex for both endonucleases. By addition of a 100-fold excess of Co-phthalocyanine complex with respect to DNA duplex the initial rate of substrate hydrolysis by restriction endonuclease SsoII is observed to decrease twice. An equimolar ratio of the metal complex and hybrid duplex leads to essentially complete inhibition of RNA cleavage by RNase H from E. coli. The inhibition of catalytic activity of enzymes recognizing the double-stranded nucleic acids in the presence of Co-phthalocyanine complex is assumed to be caused by the ability of the latter to interact with DNA, RNA, and DNA--RNA duplexes.

Base Sequence↗
PubMed1997

Chemical cross-linking of the human immunodeficiency virus type 1 Tat protein to synthetic models of the RNA recognition sequence TAR containing site-specific trisubstituted pyrophosphate analogues.

A chemical ligation procedure has been developed for the synthesis of oligoribonucleotides carrying a trisubstituted pyrophosphate (tsp) linkage in place of a single phosphodiester. Good yields of tsp were obtained when a single 2'-deoxynucleoside 5' to the tsp was used in the ligation reaction. A tsp linkage was found to be reasonably stable to hydrolysis but cleaved by treatment with ethylenediamine or lysine to give phosphoamidate adducts. A model human immunodeficiency virus type 1 (HIV-1) TAR RNA duplex containing an activated tsp was able to bind to HIV-1 Tat protein with only 3-fold reduced affinity and to a Tat peptide (residues 37-72) with identical affinity compared to that of an unmodified duplex. Tsps incorporated at sites previously identified as being in close proximity to Tat protein were able to cross-link to Tat peptide (37-72) to form a covalent phosphoamidate conjugate. Endopeptidase cleavage followed by MALDI-TOF (matrix-assisted laser desorption ionization time of flight) mass spectrometric analysis provided strong evidence that a TAR duplex containing a tsp replacing the phosphate at 38-39 had reacted specifically with Lys51 in the basic region of Tat peptide (37-72). The new chemical cross-linking method may be generally useful for identifying lysines in close proximity to phosphates in basic RNA-binding domains of proteins.

Amino Acid Sequence↗
PubMed1997

Derivatization and template-guided ligation of oligodeoxyribonucleotides using cyanogen bromide and N-substituted morpholines.

Cyanogen bromide has been found to induce the template-guided condensation of oligonucleotides only in the presence of N-substituted morpholines. Based on 31P, 1H and 13C NMR spectroscopy data, the mechanism of the phosphomonoester group activation by cyanogen bromide in N-substituted morpholine buffers is suggested. It has also been shown that BrCN can be used for the synthesis of oligonucleotide derivatives in aqueous solution.

Buffers↗
PubMed1997

Design of new reagents on the base of DNA duplexes for irreversible inhibition of transcription factor NF-kappa B.

The main purpose of the present work is to search for the optimal design of a DNA duplex containing an active group for crosslinking and irreversible inhibition of the transcription factor NF-kappa B. Modified DNA duplexes with an identical nucleotide sequence but different internucleotide phosphates replaced by the trisubstituted pyrophosphate internucleotide group were synthesized. Crosslinking of the human NF-kappa B p50 subunit with the modified DNA duplexes was carried out. It was shown that only four modified duplexes crosslinked with the NF-kappa B p50 subunit. The specificity of these reactions was confirmed. A position of the phosphate in the NF-kappa B recognition site was found where replacement on the active trisubstituted pyrophosphate group resulted in a 50% yield of crosslinking. The fact that DNA duplexes containing the trisubstituted pyrophosphate group specifically react with the NF-kappa B p50 subunit in the Escherichia coli total lysate supports the idea that such modified DNA can be used as high specific inhibitors for DNA-recognizing proteins.

Animals↗
PubMed1997

[Cyclic oligonucleotides. II. Regularities in the formation of polycyclic structures].

Cyclization of a 38-mer oligodeoxyribonucleotide on a cyclic template was studied by the chemical and enzymic ligation methods. Both structures and yields of the reaction products depended on the ligation method and the nucleotide and template sequences. The chemical ligations resulted in the formation of catenanes, whose structures were confirmed by hydrolysis with the MvaI restriction endonuclease. Presence of G/C-rich clusters near the formed internucleotide bond favored the catenane formation.

Nucleotides, Cyclic↗
PubMed1997

[Synthesis of modified oligodeoxyribonucleotides containing 9-(2'-amino-2'-deoxy-beta-D-arabinofuranosyl)adenine].

A method for preparing a modified derivative of 2'-amino-2'-deoxy-arabino-adenosine ready for directed insertion into an oligonucleotide chain during solid-phase synthesis was elaborated. A series of the title oligonucleotides (6-25-mers) containing a 2'-amino-2'-deoxy-arabino-adenosine fragment were prepared. A high reactivity of the 2'-amino group during the acylation with carboxylic acid anhydrides was demonstrated. It was shown that the insertion of the modified fragments into oligonucleotides did not inhibit the formation of the DNA duplex.

Acylation↗
PubMed1996

Crosslinking of double-stranded oligonucleotides containing O-methyl-substituted pyrophosphate groups to the HNF1 transcription factor in nuclear cell extract.

Probing of the HNF1 (hepatocyte nuclear factor I) DNA-binding region using a set of DNA duplexes containing pyrophosphate or O-methyl-substituted pyrophosphate internucleotide groups at different positions of the HNF1 recognition sequence was performed. The histidine-tagged HNF1/1-281 DNA binding domain and nuclear extract from rat liver were used. We showed that HNF1 from these species specifically binds to modified DNA duplexes. A correlation in binding affinity of both types of duplexes was detected. Crosslinking of the HNF1 DNA-binding domain and HNF1 in nuclear liver extract to DNA duplexes carrying O-methyl-substituted pyrophosphate groups was observed. The crosslinking efficiency of HNF1 in liver extract to substituted pyrophosphate-modified DNA duplex, containing a reactive internucleotide group between nucleotides G and T of the GT dinucleotide immediately 5' to the TAAT recognition sequence, amounts to 40% of the efficiency of non-covalent association. Nonspecific crosslinking of the reactive DNA duplexes to other components of nuclear extract was not observed. These results indicate that DNA duplexes carrying substituted pyrophosphate internucleotide groups can specifically bind and crosslink with DNA-binding proteins, especially transcription factors in crude preparations and could constitute a potential tool to control the expression of disease-causing genes.

Animals↗
PubMed1996

Cross-linking of SsoII restriction endonuclease to cognate and non-cognate DNAs.

Specific and non-specific interactions of SsoII restriction endonuclease (R.SsoII) were probed by the method of covalent attachment to modified DNA containing an active monosubstituted pyrophosphate internucleotide bond instead of a phosphodiester one. R.SsoII with six N-terminal His residues was shown to be cross-linked to duplexes with this type of modification, either containing or not the recognition sequence. Competition experiments with covalent attachment of R.SsoII to activated DNAs demonstrated the similar affinity of the enzyme to cognate and non-cognate DNAs in the absence of cofactor, Mg2+ ions.

Base Sequence↗
PubMed1996

NF-kappaB p50 subunit cross-linking to DNA duplexes, containing a monosubstituted pyrophosphate internucleotide bond.

The new express technique based on the use of BrCN to synthesize DNA duplexes, containing non-substituted or monosubstituted pyrophosphate internucleotide bonds has been proposed. Using this technique, DNA duplexes having modified internucleotide bonds between dT and dC residues in the human NF-kappaB transcription factor recognition sequence in HIV-1 (5'-GGAAAGTCCC-3') have been prepared. We demonstrate that these internucleotide bonds within the recognition site do not prevent the formation of NF-kappaB p50 subunit complex with the corresponding duplexes. The cross-linking of NF-kappaB p50 subunit to the DNA duplex containing a monosubstituted pyrophosphate internucleotide bond has been successfully performed.

Base Composition↗
PubMed1996

Synthesis and properties of cross-linked DNA duplexes.

A method has been devised to synthesize DNA duplexes with covalently connected strands. The structure of cross-linked duplexes was confirmed by a reaction with the restriction endonuclease AluI. The thermal stability of the resulting compounds was investigated.

Base Sequence↗
PubMed1996

[Synthesis and properties of covalently-closed DNA duplexes containing pyrophosphate and and substituted pyrophosphate internucleotide groups].

A new method for the efficient synthesis of covalently closed DNA duplexes (DNA dumbbells) and the introduction of pyrophosphate and substituted pyrophosphate internucleotide groups into their structure is proposed. The method is based on chemical ligation in DNA duplexes that are formed by a polynucleotide the ends of which are brought together due to the introduction of the minihairpin structure [sequence: see text]. DNA dumbbells containing a pyrophosphate (substituted pyrophosphate) group result from the interaction as being between the 3'-terminal phosphate (methylphosphate) group of the polynucleotide and the 5'-terminal phosphate group of deoxyguanosine of the minihairpin sequence, which flanks the polynucleotide from the 5' end. 1-Ethyl-3-(3'-dimethylaminopropyl) carbodiimide was used as a condensing agent. The yield of covalently closed 42-mer DNA duplexes containing a pyrophosphate group was 98%, that of duplexes with a substituted pyrophosphate group was 25%. The reactivity of the substituted pyrophosphate group incorporated into DNA dumbbells was studied. It is shown that the group efficiently interacts with nucleophiles in an aqueous medium at pH 8.0.

DNA↗