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Biomedical subjects

M N Repkova

Publications and source records attributed to M N Repkova.

At least 19 recordsLinked to original sources
PubMed2005

Polyclonal antibodies against a structure mimicking the covalent linkage unit between picornavirus RNA and VPg: an immunochemical study.

We propose that therapy of patients with anticancer drugs that poison DNA topoisomerases induces formation of covalent complexes of cellular RNAs and DNA topoisomerases. The appearance of these complexes can be detected with antibodies against a synthetic hapten mimicking the covalent linkage unit Tyr-pU(p) of picornavirus RNA and VPg. We synthesized hapten [N(Ac),CO(NH2)]Tyr-(5 P --> O)Up-O-(CH2)6NH2, conjugated it with BSA, and immunized rabbits with the antigen obtained. The raised polyclonal antibodies were purified by successive affinity chromatography on BSA-Sepharose and hapten-Sepharose columns. Target antibodies recognized hapten and encephalomyocarditis virus RNA-VPg complex specifically as found using the dot-immunogold method. We believe that these antibodies might be useful to study mechanism of picorna and similar virus RNA synthesis. The discovery and qualitative determination of the cellular RNA-DNA topoisomerases covalent complexes with these antibodies might be useful to monitor therapy efficacy by drugs "freezing" dead-end complexes of DNA topoisomerases and nucleic acids and to understand the mechanism of DNA topoisomerase poisoning in situ.

Animals↗
PubMed2005

2'-bis-pyrene modified oligonucleotides: sensitive fluorescent probes of nucleic acids structure.

A new type of fluorescent nucleic acid probes, 2-bis-pyrene-modified oligonucleotides, is described. Preparation of these conjugates involves attachment of two pyrene moieties to the 2'-phosphate group introduced into any position within a sequence by solid-phase phosphoramidite synthesis. Good hybridization properties of the 2'-bis-pyrene probes, their nuclease resistance and sensitivity of fluorescence to the type of complementary nucleic acid have been demonstrated.

DNA↗
PubMed2004

Silencing of MDR 1 gene in cancer cells by siRNA.

Inhibition of p-glycoprotein (PGP) expression and reverse of multidrug resistance (MDR) phenotype in KB-8-5 cells by synthetic 21-bp double-stranded oligoribonucleotides were investigated. siRNA constructs for the efficient down regulation of MDR1 that are active in nanomolar concentrations and cause reversal of MDR phenotype in cells were developed.

Base Sequence↗
PubMed2004

Two-component 10-23 DNA enzymes.

A new strategy for engineering of catalytic two-component constructions based on 10-23 DNAzyme was proposed. The using of a combination of shortened DNAzyme with 2'-O-methyl oligomers as effectors significantly increased the catalytic activity of this DNAzyme.

Base Sequence↗
PubMed2000

[Cleavage of RNA in hybrid duplexes by E. coli ribonuclease H. II. Substrate properties of nucleotides containing non-nucleotide linkers].

The 20-mer bridged oligodeoxynucleotides containing short oligomers joined by the hexamethylenediol and hexaethylene glycol linkers were shown to form complementary DNA/DNA and RNA/DNA complexes whose thermostability depends on the length and number of the nonnucleotide linkers. Hybrid complexes of the bridged oligonucleotides proved to be substrates for the E. coli ribonuclease H. The presence of one-three nonnucleotide linkers in a 20-mer decreased the hydrolysis efficacy only 1.2-1.4-fold. It is the composition of the RNA cleavage products that was influenced the most significantly by the nonnucleotide linkers. RNase H simultaneously hydrolyzed the RNA 3'-ends of each hybrid duplex involving a bridged oligonucleotide. The presence of an inverted 3'-3'-phosphodiester bond at the 3'-end of the oligodeoxyribonucleotide only slightly affected the RNase H activity.

DNA↗
PubMed1999

Proteins neighboring 18S rRNA conserved sequences 609-618 and 1047-1061 within the 40S human ribosomal subunit.

The structure of human 40S ribosomal subunits has been probed by a cross-linking strategy based on the use of oligonucleotide derivatives that modify proteins in the vicinity of specific 18S rRNA sequences. The oligonucleotide derivatives carried a p-azidoperfluorobenzamide group at the 5' ends and were complementary to 18S rRNA sequences 609-618 and 1047-1061, homologous to the highly conserved regions designated as the "530 stem-loop" and "790 stem-loop", respectively, in Escherichia coli 16S rRNA. Ribosomal proteins surrounding these sequences were the main targets of the cross-linking. Proteins S3 and S5 were cross-linked to the derivative complementary to the sequence 609-618, and proteins S2 and S3 were modified by the derivative complementary to the sequence 1047-1061. Cross-linking was not affected by binding of 40S subunits to either poly(U) or poly(U) and Phe-tRNA(Phe).

Base Sequence↗
PubMed1997

[Artificial ribonucleases I. Targeted RNA cleavage by 5'-peptidyloligodeoxyribonucleotides containing arginine and leucine residues].

The interaction of DNA and RNA with oligodeoxyribonucleotides and their 3'-terminal N-(2-hydroxyethyl)phenazinium derivatives carrying peptide residues with alternating basic and hydrophobic amino acids at the 5'-terminal phosphate was studied. It was found that the introduction of peptide residues (LeuArg)n-Gly-NH2 (n = 2-4) into an oligodeoxyribonucleotide enhances the latter's hybridization ability: each additional LeuArg pair increases the Tm value of the (5')pd(CACACACAAAAAAC).(3')d(TGTGTGTG)p(-LeuArg)n-Gly- NH2 complex by 1.3 degrees C. The reagents did not destort the DNA structure and were capable of site-specific hydrolysis of the phosphodiester bonds of RNA. It was shown that the location of the cleavage sites and the efficacy of the RNA hydrolysis at n = 2 and 4 and at n = 3 strongly differ. The maximum hydrolysis (80%) of tetradecaribonucleotide (5')p(GAUUGAAAAUCCCC) was achieved using peptidyloligodeoxyribonucleotide (3')d(CTAACT)p(LeuArg)4GlyNH2. The possibility of directed cleavage of phosphodiester bonds in tRNAPhe by peptidyloligodeoxyribonucleotides (3')d(CTAACT)p(LeuArg)nGlyNH2 (n = 3 and 4) was shown.

Arginine↗
PubMed1997

Nucleotide G-1207 of 18S rRNA is an essential component of the human 80S ribosomal decoding center.

mRNA analogues-derivatives of oligoribonucleotides consisting of two different codons and bearing an aryl azide group at the 5'-phosphates-were crosslinked to human 80S ribosomes by UV-irradiation of the various model complexes obtained in the presence of the cognate tRNAs. Three sequences, namely pUUUGUU (coding for Phe and Val), pUUCUAAA (first triplet coding for Phe and second being stop-codon), and pGUGUUU (coding for Val and Phe), have been used. Sequences of 18S rRNA containing nucleotides crosslinked to the mRNA analogues were examined by hydrolysis with RNase H in the presence of various cDNA probes. Crosslinked nucleotides were identified by primer extension. In all cases, only nucleotide G-1207 (equivalent to G-926 in Escherichia coli 16S rRNA) has been detected as crosslinked. Crosslinking of the mRNA analogues to the large ribosomal subunit was negligible.

Benzoates↗
PubMed1995

Interaction of primer tRNA(Lys3) with the p51 subunit of human immunodeficiency virus type 1 reverse transcriptase: a possible role in enzyme activation.

In the interaction between HIV-1 RT and tRNA(Lys3) each subunit of the heterodimer interacts with tRNA showing a different affinity: Kd (p66) = 23 nM, Kd (p51) = 140 nM. Preincubation of heterodimeric RT with tRNA, at concentrations similar to that of the Kd value for p51, leads to an increase of the catalytic activity on poly(A)-oligo(dT). These results were compared to those using different tRNA analogs: oxidized tRNA, tRNAs lacking one, two or three nucleotides from the 3'-end, or ribo- and deoxyribonucleotides mimicking the anticodon loop sequence. In all cases, tRNA analogs were weaker activators of HIV-1 RT than natural tRNA. A possible mechanism of RT p66/p51 activation by tRNA and its analogs, mediated through the p51 subunit, is discussed.

Anticodon↗
PubMed1994

The solution structure of a 3'-phenazinium (Pzn) tethered DNA-RNA duplex with a dangling adenosine: r(5'G-AUUGAA3'):d(5'TCAATC3'-Pzn).

The 3'-Pzn group tethered to an oligo-DNA stabilizes a DNA-RNA hybrid duplex structure by 13 degrees C compared to the natural counterpart. This report constitutes the first full study of the conformational features of a hybrid DNA-RNA duplex, which has been possible because of the unique stabilization of this rather small duplex by the tethered 3'-Pzn moiety (Tm approximately 40 degrees C from NMR). In this study, a total of 252 inter- and intra-strand torsional and distance constraints along with the full NOE relaxation matrix, taking into account the exchange process of imino and amino protons with water, have been used. The 3'-Pzn-promoted stabilization of the DNA-RNA hybrid duplex results in detailed local conformational characteristics such as the torsion angles of the backbone and sugar moieties that are close to the features of the other natural DNA-RNA hybrids (i.e. sugars of the RNA strand are 3'-endo, but the sugars of the DNA strand are intermediate between A- and B-forms of DNA, 72 degrees < P < 180 degrees; note however, that the sugars of our DNA strand have a C1-exo conformation: 131 degrees < P < 154 degrees). This study suggests that 3'-Pzn-tethered smaller oligo-DNA should serve the same purpose as a larger oligo-DNA as a antisense inhibitor of the viral mRNA. Additionally, these types of tethered oligos have been found to be relatively more resistant to the cellular nuclease. Moreover, they are taken up quite readily through the cellular membrane (14) compared to the natural counterparts.

Hydrogen Bonding↗
PubMed1994

Alterations at the 3'-CCA end of Escherichia coli and Thermus thermophilus tRNA(Phe) do not abolish their acceptor activity.

The 3'-CCA end of tRNA(Phe) from Escherichia coli and Thermus thermophilus was changed to AAA, CCC, UUU and UUA by the stepwise degradation procedure of the 3'-CCA end of tRNA(Phe) followed by the ligation with oligoribonucleotides. Substrate activity of tRNA(UUAPhe) and tRNA(CCCPhe) in tRNA aminoacylation was shown. tRNA(AAAPhe) is a bad substrate for E. coli and Th. thermophilus phenylalanyl-tRNA synthetases. tRNA(UUUPhe) has no detectable activity in tRNA aminoacylation. Therefore the nature of the 3'-end of tRNA(Phe) plays an important role in tRNA binding and its substrate efficiency. Nevertheless the CCA sequence at the 3'-end of tRNA(Phe) does not seem to be an absolute requirement for tRNA aminoacylation.

Escherichia coli↗
PubMed1994

Effect of derivatization of ribophosphate backbone and terminal ribophosphate groups in oligoribonucleotides on their stability and interaction with eukaryotic cells.

Various derivatives of oligoribonucleotides were synthesized by the H-phosphonate method. Different modifications of the ribophosphate backbone were designed in order to protect the derivatives against nucleolytic enzymes present in the biological media. These modifications include coupling of fluorescein moiety to 3'-terminal ribose, 2'-O-methylation of ribose, introduction of phosphorothioate internucleotide bonds throughout the molecule, replacement of the two last 3'-terminal phosphodiester bonds by phosphoroamidates and coupling of the last 3'-terminal nucleotide via the 3'-3'-phosphodiester bond. All modifications were tested for their effect on the stability of the derivatives against phosphodiesterase from snake venom and nucleases of the cell culture media. 2'-O-methylated oligoribonucleotides containing either terminal 3'-3'-linkage or two 3'-terminal phosphoroamidate internucleotide bonds appeared to be the most stable under the most severe conditions used. The results demonstrate a possibility to use protected oligoribonucleotide derivatives for experiments in vivo when the use of deoxy-analogues might be ineffective. The uptake of 2'-O-methylated derivatives and their 5'-cholesterol conjugates (coupled via a disulfide bond) by human carcinoma cells did not differ from that of the corresponding oligodeoxyribonucleotides. 85% of the bound derivatives were found in the membrane-cytosolic fraction, while only 15% were found in the nuclear fraction. The oligonucleotide moiety of 2'-O-methyloligoribonucleotide-cholesterol conjugate was not translocated through the cellular membrane. After cleavage of the linkage between cholesterol and oligonucleotide by dithiothreitol the major portion of the oligonucleotide moiety was released into the media. The derivatives, as well as their 5'-cholesterol conjugates, which entered the cells, were stable and protected from action of dithiothreitol dissolved in culture media. These results demonstrate an endocytosis mechanism of penetration as observed in similar experiments using oligodeoxyribonucleotides.

Animals↗
PubMed1994

[The role of the 3'-CCA sequence in the interaction of tRNA(Phe) from E. coli and Thermus thermophilus with homologous phenylalanyl-tRNA-synthetases].

The 3'-CCA end of tRNA(Phe) from E. coli and Thermus thermophilus was modified by stepwise degradation and ligation of the shortened tRNA with different trinucleotides (pUpUpA, (pA)3, (pC)3, (pU)3). Kinetic parameters for the aminoacylation reaction of modified tRNAs have been determined. The role of the 3'-terminal trinucleotide of tRNA(Phe) in tRNA binding and aminoacylation by phenylalanyl-tRNA synthetases from E. coli and Thermus thermophilus is postulated.

Acylation↗
PubMed1993

Structural arrangement of the codon-anticodon interaction area in human placenta ribosomes. Affinity labelling of the 40S subunits by derivatives of oligoribonucleotides containing the AUG codon.

Using the derivatives of the oligoribonucleotides pAUGUn and AUGUnC (n = 0; 3) bearing an alkylating group at either the 5' or 3' end, respectively (mRNA analogues), the affinity labelling of the human placenta 40S ribosomal subunits has been investigated in model initiation complexes obtained in the presence of the ternary complex eIF-2.GTP.Met-tRNA(fMet). The regions of 18S rRNA and ribosomal proteins labelled with these mRNA analogues were identified. The sites of covalent attachment of the pAUGUn derivatives with a reactive group at the 5' end were located between 18S rRNA positions 976 and 1164. The derivative of AUGU3C with an alkylating group at the 3' end modified 18S rRNA mainly at the 593-673 region. The main targets of the 3' end derivative of AUGC were located between positions 1610 and 1869. The proteins S3/S3a, S6, S7 and S14/S15 were modified by both types of the oligoribonucleotide derivatives regardless of the point of the reactive group attachment to the oligonucleotide moiety. The proteins S2 and S4 were modified by both the 3' end derivative of AUGC and 5' end derivative of pAUGU3; and the protein S8 was modified by the 3' end derivative of AUGC. The proteins S5 and S9 were labelled by the 5' end derivative of pAUGU3, and the protein S17 was modified by the 5' end derivative of pAUG.

Affinity Labels↗
PubMed1993

Photomodification of RNA and DNA fragments by oligonucleotide reagents bearing arylazide groups.

Photomodification of ribo- and deoxyribo-octanucleotides by oligonucleotide reagents (6- and 7-mers) bearing p-azidotetrafluorobenzamido and 2-nitro-5-azidobenzamido groups has been investigated. It is shown that the oligonucleotides with a perfluoroarylazide group were effective modifiers both of deoxyribo- and ribo-targets. Maximum extent of cross-linked product formation (70%) was obtained when the deoxyribo-octanucleotide was modified by a heptanucleotide reagent with a perfluoroarylazide group. Selectivity of the photomodification was also high (50% on the G-residue at a certain position).

Azides↗
PubMed1993

[Affinity modification of 40S ribosomal subparticles from human placenta with mRNA analogs--AUGUnC oligoribonucleotide analogs with an alkylating group at the 3'-end].

Using 2',3'-O-[4-N-(2-chloroethyl)-N-methylamino]benzylidene derivatives of AUGUn[32P]pC (mRNA analogues), affinity labelling of human placenta 40S ribosomal subunits has been investigated in model initiation complexes obtained in the presence of the ternary complex eIF-2.GTP.Met-tRNA(fMet). The regions of 18S rRNA labelled with these mRNA analogues were identified. The main targets of 18S rRNA alkylation by the derivative of AUG[32P]pC were located within positions 1610-1747 and 1748-1869. The site of covalent attachment of AUGU3[32P]pC derivative to 18S rRNA was found within positions 593-673. Taking into account the data on labelling of human placenta ribosomes with the same derivatives of oligourydilates obtained previously, the conclusion was made that the arrangement of the codon U3 in the mRNA-binding centre of the initiation complex 40S.AUGU3[32P]pC derivative.eIF-2.GTP.Met-TPHK(fMet) differs from the arrangement of the same codon at the A-site of the complex imitating the pretranslocation state of ribosomes.

Affinity Labels↗