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At least 19 recordsLinked to original sources

PubMed2006

Solution structure of DNA/RNA hybrid duplex with C8-propynyl 2'-deoxyadenosine modifications: Implication of RNase H and DNA/RNA duplex interaction.

Solution structures of DNA/RNA hybrid duplexes, d(GCGCA*AA*ACGCG): r(cgcguuuugcg)d(C) (designated PP57), containing two C8-propynyl 2'-deoxyadenosines (A*) and unmodified hybrid (designated U4A4) are solved. The C8-propynyl groups on 2'-deoxyadenosine perturb the local structure of the hybrid duplex, but overall the structure is similar to that of canonical DNA/RNA hybrid duplex except that Hoogsteen hydrogen bondings between A* and U result in lower thermal stability. RNase H is known to cleave RNA only in DNA/RNA hybrid duplexes. Minor groove widths of hybrid duplexes, sugar puckerings of DNA are reported to be responsible for RNase H mediated cleavage, but structural requirements for RNase H mediated cleavage still remain elusive. Despite the presence of bulky propynyl groups of PP57 in the minor groove and greater flexibility, the PP57 is an RNase H substrate. To provide an insight on the interactions between RNase H and substrates we have modeled Bacillus halodurans RNase H-PP57 complex, our NMR structure and modeling study suggest that the residue Gly(15) and Asn(16) of the loop residues between first beta sheet and second beta sheet of RNase HI of Escherichia coli might participate in substrate binding.

DNA↗

PubMed1995

Kinetic analysis of Escherichia coli ribonuclease HI using oligomeric DNA/RNA substrates suggests an alternative mechanism for the interaction between the enzyme and the substrate.

Escherichia coli ribonuclease HI mainly recognizes the DNA/RNA hybrid regions preceding the cleavage site. To understand the interaction between the enzyme and the substrate in more detail, the kinetic properties of the enzyme, as well as its variant with mutations in the basic protrusion, were studied using a series of oligomeric DNA/RNA hybrids as substrates. These substrates were prepared by hybridizing a 12-b RNA (5'-CGGAGAUGACGG-3') with DNA oligomers varying in size and sequence. The 12-b RNA hybridized to the complementary 12-b DNA was primarily cleaved at A9-C10. Since an increase in the length of the RNA between the cleavage site and the 5' end of the DNA/RNA hybrid, achieved using a longer DNA/RNA substrate, did not seriously affect the kinetic parameters of the enzyme, the 12-bp DNA/RNA hybrid seems to be large enough to contact the entire substrate-binding site of the enzyme. The kinetic data presented here suggest that the DNA residues complementary to the RNA residues located six or seven residues upstream from the cleavage site interact with the basic protrusion of the enzyme, regardless of whether or not it is hybridized to the RNA strand. Such an interaction is permitted only when the conformation of either the enzyme or the substrate, or both, is changed upon binding.

Base Sequence↗

PubMed2001

Solution structure of an arabinonucleic acid (ANA)/RNA duplex in a chimeric hairpin: comparison with 2'-fluoro-ANA/RNA and DNA/RNA hybrids.

Hybrids of RNA and arabinonucleic acid (ANA) as well as the 2'-fluoro-ANA analog (2'F-ANA) were recently shown to be substrates of the enzyme RNase H. Although RNase H binds to double-stranded RNA, no cleavage occurs with such duplexes. Therefore, knowledge of the structure of ANA/RNA hybrids may prove helpful in the design of future antisense oligonucleotide analogs. In this study, we have determined the NMR solution structures of ANA/RNA and DNA/RNA hairpin duplexes and compared them to the recently published structure of a 2'F-ANA/RNA hairpin duplex. We demonstrate here that the sugars of RNA nucleotides of the ANA/RNA hairpin stem adopt the C3'-endo (north, A-form) conformation, whereas those of the ANA strand adopt a 'rigid' O4'-endo (east) sugar pucker. The DNA strand of the DNA/RNA hairpin stem is flexible, but the average DNA/RNA hairpin structural parameters are close to the ANA/RNA and 2'F-ANA/RNA hairpin parameters. The minor groove width of ANA/RNA, 2'F-ANA/RNA and DNA/RNA helices is 9.0 +/- 0.5 A, a value that is intermediate between that of A- and B-form duplexes. These results rationalize the ability of ANA/RNA and 2'F-ANA/RNA hybrids to elicit RNase H activity.

Arabinonucleotides↗

PubMed2001

In vivo and in vitro influence of selenium on DNA/RNA synthesis in spleen and lymphocytes in culture--possible mediation of changes in GSH/GSSG ratio.

Effect of superanutritional levels of selenium (Se) as sodium selenite (0.5 and 1.5 ppm) given orally to Balb/c mice for one and two weeks was observed on the rate of DNA/RNA synthesis, levels of reduced as well as oxidized glutathione (GSH and GSSG) and glutathione peroxidase (GSH-Px)/glutathione-S-transferase (GSH-S-transferase) activities in spleen. Similar effect of three different concentrations of Se (10(-7), 10(-5) and 10(-3) M) in culture media was also observed on the rate of DNA/RNA synthesis in proliferating lymphocytes taken from mice spleen. The results of the present study indicated that with increasing concentration and duration of Se treatment in vivo and in vitro, a marked inhibition of the rate of DNA/RNA synthesis was observed. Levels of total glutathione and GSSG in spleen were elevated significantly only after two weeks in 1.5 ppm treatments. Glutathione peroxidase activities in spleen decreased (p < 0.05) in 1.5 ppm group at one week and in 0.5 ppm group at two week treatment. At higher Se treatment, the activity recovered towards control. However, GSH-S-transferase in spleen remained unchanged at all treatment intervals. The results indicated that changes in glutathione system by increasing Se concentration might account for inhibition of rate of DNA/RNA synthesis.

Animals↗

PubMed1999

Influence of sodium selenite and selenomethionine on DNA/RNA synthesis and BaP binding to spleen lymphocytes in culture.

In the present study, an attempt was made to provide some information regarding the effects of organic and inorganic selenium compounds on DNA/RNA synthesis and benzo(a)pyrene uptake in cultured lymphocytes from mice spleen. It was clear from the results that there was a significant inhibition of DNA/RNA synthesis with increasing concentration of either form of selenium from 0.1 microM to 1 mM in culture medium. However, when used at the same level as selenite with respect to selenium content, selenomethionine exerted more DNA/RNA synthesis inhibitory effect than selenite. Benzo(a)pyrene uptake by proliferating lymphocytes was also significantly reduced with increasing selenium concentration (0.1 microM to 1 mM). However, both forms of selenium at the same selenium concentration showed almost the same inhibitory effect on the cellular uptake of benzo(a)pyrene, which indicated that some factor(s) other than the DNA synthesis are also involved in the interaction between benzo(a)pyrene and cells. Involvement of the changes in the carcinogen metabolism and glutathione level has been discussed. Present studies show that organic selenium as a source of selenium is a more potent chemopreventive compared to the inorganic one. This information may have a useful therapeutic potential.

Animals↗

PubMed2005

Origin and evolution of viruses: escaped DNA/RNA sequences as evolutionary accelerators and natural biological weapons.

Knowledge of the origin and evolution of viruses could provide a better understanding of a number of phenomena in the field of evolution such as the origin and development of multi-cellular organisms, the rapid diversification of species over the last 600-700 million years and the lack of transitional forms in the evolution of species ("missing links") etc. One of the possible effects of escaped DNA/RNA sequences or viruses on the evolution of multi-cellular organisms, especially vertebrates, could be the phenomenon of horizontal transmission and dissemination of genes. Interestingly, if so, this effect could be considered as a model of primeval and natural genetic engineering. Other possible links between the evolution of multi-cellular organisms and viruses are connected with the fact that viruses represent the source of different forms of selective pressure such as epidemics of infectious diseases, autoimmunity, malignant alteration, reproductive efficiency, etc. At the same time, these two models of "long-term evolutionary relations" could represent "key factors" in the evolution between viruses and multi-cellular organisms. The capability of a genome to produce and emit DNA/RNA sequences or de novo created viruses which can be a vector of genes horizontal transmission and/or cause selective pressure on concurrent or predator species gives a new characteristic to viruses--the possibility of their acting as natural biological weapons. Finally, possibly evolutionary advantages of this genome capability could be one of explanations for the phenomena such as genome instability and its ability to emit DNA/RNA sequences and/or de novo created viruses, as well as evolutionary conservation of this unique phenomena.

Animals↗

PubMed2000

Ribonuclease H attack of leukaemic fused transcripts AML1-MTG8 (ETO) by DNA/RNA chimeric hammerhead ribozymes.

BACKGROUND: Catalytic anti-sense oligonucleotides might be useful tools for controlling specific gene expression. However, to obtain effective oligonucleotides of the desired function in vivo is still a difficult task. RESULTS: To evaluate the usefulness of synthesized DNA/RNA hammerhead ribozymes targeting AML1-MTG8 (ETO) leukaemic fusion transcripts in vivo, we analysed their effects on cell growth and the mechanism of action using isolated cell nuclei. These ribozymes inhibited the growth of leukaemic cell lines expressing the AML1 -MTG8 and degraded AML1-MTG8 mRNA in isolated nuclei of these cells. However, the reactions gave rise to additional cleavage products. Systematic cleavage analyses using an anti-sense oligonucleotide array revealed that the cleavage was induced by endogenous RNase H at specific sites, in accordance with their calculated melting temperature (Tm) values. With suppression of RNase H by sulfhydryl agents, the DNA/RNA ribozyme had a ribozyme catalytic activity. In addition, the ribozymes and anti-sense oligonucleotides suppressed the AML1-MTG8 protein in the leukaemic cells. CONCLUSIONS: The DNA/RNA ribozymes inhibited cell growth primarily via anti-sense effects, the main role of which was the activation of RNase H-digestion by their DNA arms. In addition, the isolated nuclei provided a useful assay system for modelling in vivo conditions for the quantitative evaluation of anti-sense/ribozyme activity.

Antisense Elements (Genetics)↗

PubMed2001

Characterization of DNA, RNA and DNA/RNA duplexes containing an L-nucleotide.

In order to investigate the duplex structure of DNA and RNA containing an L-nucleotide residue, we carried out the UV-melting and CD experiments. Although the introduction of an L-nucleotide into DNA/DNA, DNA/RNA and RNA/RNA duplexes reduced their Tm values, the typical two-state transitions were observed for all of the duplexes. The incorporation of an L-nucleotide into the RNA strand caused more remarkable decreases of the Tm value than that into the DNA strand. The CD spectra of the DNA/DNA and RNA/RNA duplexes containing an L-nucleotide are similar to those of unmodified duplexes. On the other hand, the spectra of the heterochiral DNA/RNA hybrid duplexes are significantly different from those of the homochiral DNA/RNA hybrid duplexes, which are generally thought to adopt the A-form.

Base Sequence↗

PubMed2003

Structure of a B-form DNA/RNA chimera (dC)(rG)d(ATCG) complexed with daunomycin at 1.5 A resolution.

The crystal structure of a DNA/RNA chimera (dC)(rG)d(ATCG) complexed with the anticancer drug daunomycin has been determined at 1.5 A resolution with R(work) and R(free) of 19.7 and 23.3%, respectively, for 2767 reflections. The complex crystallizes in space group P4(1)2(1)2, with unit-cell parameters a = b = 28.05, c = 53.16 A, and contains one nucleic acid strand and one daunomycin molecule in the asymmetric unit. To our knowledge, this is the first crystal structure of a DNA/RNA chimera complexed with an intercalating drug. The DNA/RNA chimera adopts the B-form helical conformation, with the 2'-hydroxyl group in the major groove of the duplex, forming hydrogen bonds to N7 and the anionic phosphate oxygen of its 3'-side adenine. The present results indicate that the replacement by the ribose sugar in the DNA sequence does not change the geometry and intercalation pattern of daunomycin. A model of B-form RNA has been built based on the present structure. The model indicates that the interactions of the 2'-hydroxyl groups in the B-form duplex depend on their 3'-side nucleotides.

Antibiotics, Antineoplastic↗

PubMed1996

Profile of cell cycle in hematopoietic malignancy by DNA/RNA quantitation using 7AAD/PY.

Using 7-amino-actinomycin-D/pyronin Y (7AAD/PY), we analyzed the surface phenotypes and cell cycle of 22 hematopoietic cell lines based on their cellular DNA/RNA content. Populations of G1a, G1b, S, and G2M, the DNA index (DI), and the RNA index of S phase (SRI) were calculated by means of DNA/RNA dot plots. Two new parameters were extracted from the cell-cycle profiles: the nucleic acid index of S phase (NI) and the coefficient of variations in the RNA at S phase (SVC). DNA/RNA dot plots of cell lines revealed four characteristic profiles of the cell cycle, defined with the calculated NI and SCV. These were type 0 (small NI, large SCV), type I (small NI, small SCV), type II (large NI, small SCV), and type III (large NI, large SCV). Type O included four stem cell lines: one t(1;19) leukemia, two Ph1+ acute lymphocytic leukemia (ALL), and one biphenotypic crisis of chronic granulocytic leukemia (CGL). Type I included five ALL cell lines: three T-ALL and two common B-ALL. Type II contained 10 myeloid cell lines: five AML and five myeloid crisis of CGL. Type III contained three relatively immature lymphoma cell lines: two Burkitt's lymphoma and one follicular center lymphoma. Calculated NI/SCV (%) were as follows: type 0, 2.27 +/- 0.19/16.7 +/- 3.7; type I, 2.20 +/- 0.30/11.1 +/- 0.7; type II, 3.64 +/- 0.52/11.8 +/- 1.0; and type III, 3.60 +/- 0.53/17.5 +/- 1.9. Cell-cycle analysis of blasts using 7AAD/PY combined with surface phenotyping may yield important information for classifying hematopoietic malignancy within 2 hours of patient admission.

Antigens, CD↗

PubMed2006

An integrated microfluidic chip for DNA/RNA amplification, electrophoresis separation and on-line optical detection.

This study presents an integrated microfluidic chip capable of performing DNA/RNA (deoxyribonucleic acid/ribonucleic acid) amplification, electrokinetic sample injection and separation, and on-line optical detection of nucleic acid products in an automatic mode. In the proposed device, DNA/RNA samples are first replicated using a micromachine-based PCR module or reverse transcription PCR (RT-PCR) module and then transported by a pneumatic micropump to a sample reservoir. The samples are subsequently driven electrokinetically into a microchannel, where they are separated electrophoretically and then detected optically by a buried optical fiber. The various modules of the integrated microfluidic chip are fabricated from cheap bio-compatible materials, such as PDMS, polymethylmethacrylate, and soda-lime glass. The functionality of the proposed device is demonstrated through its successful application to the DNA-based bacterial detection of Streptococcus pneumoniae and the RNA-based detection of Dengue-2 virus. It is shown that the low thermal inertia of the PCR/RT-PCR modules reduces the sample and reagent consumption and shortens the reaction time. With less human intervention, the subsequent DNA separation and detection could be performed in an automatic mode. The integrated microfluidic device proposed in this study represents a crucial contribution to the fields of molecular biology, genetic analysis, infectious disease detection, and other biomedical applications.

DNA↗

PubMed1996

Designing of chimeric DNA/RNA hammerhead ribozymes to be targeted against AML1/MTG8 mRNA.

For therapeutic purposes, two chimeric DNA/RNA hammerhead ribozymes were synthesized to cleave AML1/MTG8, the t(8;21)-associated fusion mRNA of acute myeloid leukemia. One ribozyme, A/MRZ-1, recognizes the area adjacent to the fusion point between AML1 and MTG8, and cleaves six bases downstream from this point. The other, MRZ-1, recognizes the MTG8 sequence. Both ribozymes cleaved synthetic chimeric DNA/RNA substrates at theoretical sites. Neither cleaved AML1 RNA. A/MRZ-1 cleaved only AML1/MTG8 RNA, and MRZ-1 cleaved both AML1/MTG8 and MTG8 RNAs. The two ribozymes showed growth inhibition of an acute myeloid leukemia cell line carrying t(8;21), SKNO-1 cells. The same extent of growth inhibition was attained by antisense oligonucleotides against AML1/MTG8 RNA. The results suggest that the ribozyme has the potential to be developed as a useful agent for gene therapy, in particular for leukemia with t(8;21).

Antineoplastic Agents↗

PubMed1990

Effects of growth phase and antibiotics on quantitative DNA/RNA hybridization.

Synthetic probes complementary to ribosomal RNA are increasingly used in the detection of bacteria. Many applications, however, require the quantitation of bacteria. We therefore tested the influence of growth phase and representative antibiotics (ampicillin, chloramphenicol and gentamycin) on the outcome of DNA/RNA filter hybridization using radiolabelled probes and a multisample digital autoradiograph for quantitative monitoring. Hybridization efficiency seemed influenced by the binding capacity of the membrane, availability of target molecules and physiological growth control. For chloramphenicol the absolute hybridization signal remained constant over the experimental period. Only a slight decrease was found in experiments with gentamycin whereas viable counts dropped 10,000-fold. For ampicillin a decrease in viable counts was paralleled by diminishing signal strengths. Relative signal strengths (counts per viable cell) increased in all experiments with antibiotics. In conclusion; (i) RNA probes seem to detect bacteria even after onset of antimicrobial therapy; (ii) DNA/RNA filter hybridization appears not suitable for accurate quantitation of bacteria.

Anti-Bacterial Agents↗

PubMed2006

The dicationic derivatives of DBTAA: Interactions with DNA/RNA and antiproliferative effects on human cell lines.

Three dibenzotetraaza[14]annulenes non-covalently interacted with double-stranded DNA and RNA by mixed minor groove and/or intercalative binding mode. Observed interactions were strongly dependent on the steric exposure of positive charges and the length of the linkers of studied compounds as well as on the secondary structure and basepair composition of DNA/RNA. Compound 2 showed pronounced selectivity toward dA-dT-rich sequences and binding mode switch from dominant minor groove binding to ds-DNA to dominant intercalation into ds-RNA. Antiproliferative effect of studied compounds on human tumor and normal cell lines was in good agreement with the strength of observed interactions with DNA/RNA.

Antineoplastic Agents↗

PubMed2005

Lytic enzyme, labiase for a broad range of Gram-positive bacteria and its application to analyze functional DNA/RNA.

The lytic activity of labiase and achromopeptidase for bacterial DNA/RNA extraction were compared. Rapid lysis of many bacterial strains was observed with labiase followed by SDS treatment. Both labiase and achromopeptidase showed high lytic activity against bacterial strains with the A1alpha chemotype (e.g., Aerococcus viridans) and the A3alpha chemotype (e.g., Staphylococcus epidermidis) for cell wall peptidoglycan structures. The lytic activity of labiase was higher than that of achromopeptidase against strains with the A1gamma chemotype (e.g., Bacillus subtilis). The activity of labiase was not detrimentally affected with increasing NaCl concentration. Labiase lysates were successfully used for rapid extraction of DNA and RNA, whereas achromopeptidase lysates degraded RNA. The DNA and RNA obtained were successfully used for 16S rRNA amplification and real-time RT-PCR detection. It is concluded that labiase is useful for rapid lysis of a wide variety of Gram-positive bacteria and can be used for DNA/RNA isolation protocols.

Bacterial Outer Membrane Proteins↗

PubMed1994

Effects of arabinosylcytosine-substituted DNA on DNA/RNA hybrid stability and transcription by T7 RNA polymerase.

Cytosine arabinoside (araC) is a potent antileukemic agent which interferes with DNA replication both as a dNTP competitive inhibitor as well as after its misincorporation into DNA. We previously developed a chemical methodology for the synthesis of DNA oligomers containing araC which allowed us to study its site specific effects on duplex stability and chemical reactivity [Beardsley, G. P., Mikita, T., Klaus, M., & Nussbaum, A. (1988) Nucleic Acids Res. 16, 9165], as well as its effects on DNA ligase and DNA polymerase activity [Mikita, T., & Beardsley, G. P. (1988) Biochemistry 27, 4698]. The DNA polymerase studies, in addition to other observations, showed that araC in DNA templates could have an inhibitory effect on polymerase bypass. As a template lesion, there exists the potential for interference with other aspects of DNA metabolism, such as transcription. We have characterized a DNA/RNA hybrid containing an araC-G base pair, comparing thermal stability, chemical cleavage rates, and duplex gel mobility to an identically sequenced DNA duplex. We find that the A-form DNA/RNA hybrid and the B-form DNA duplex are nearly identical in the extent their thermal stability is affected by an araC-G(dG) base pair. Substitutions of araC for dC were made at various positions in a series of DNA duplex substrates containing a T7 RNA polymerase promoter with variable length coding strands. These were used to probe the effect of araC on promoter recognition, initiation, and elongation by T7 RNA polymerase in vitro. Substitutions in the central promoter region had no observable effect on RNA polymerase binding, initiation rate, or transcriptional output. Coding strand substitutions defined an area of high sensitivity in the initiation region where miss-starts, primer slippage, and an inability to escape from abortive cycling occur depending on the position substituted. Substitutions after position 10 had little effect on transcription output. These highly variable, position dependent effects indicate a narrow window of vulnerability where transcription output is severely reduced (approximately 100-fold) by a subtle DNA lesion that has little or no consequence when situated elsewhere in these small coding units.

Base Sequence↗

PubMed1990

Mixed DNA/RNA polymers are cleaved by the hammerhead ribozyme.

A series of chemically synthesized oligodeoxyribonucleotides containing one or two ribonucleotides (DNA/RNA mixed polymers) at and/or adjacent to the cleavage site of the substrate can be cleaved by the "hammerhead" ribozyme. In comparison with the all-RNA substrate, the predominantly deoxyribonucleotide substrates have (1) lower optimal temperatures of cleavage, (2) approximately 6-fold higher Km's and 7-fold lower kcat's at 30 degrees C, and (3) 15-fold higher Km's and 8-fold lower kcat's at 37 degrees C. The extent to which the RNA substrate cleavage is inhibited in the presence of an all-DNA (KI = 13 microM) and an RNA substrate analogue with a dC at the cleavage site (KI = 0.96 microM) supports the contention that the formation of the ribozyme-substrate complex with the predominantly deoxyribonucleotide substrates (D substrates) is impaired. The weaker binding of D substrates was confirmed by thermal denaturation and determination of the Tm of the complex. Analysis of the kinetic data also suggests that the conformation of the catalytic core of the ribozyme-substrate complex differs from that of the all-RNA complex, a change that results from the presence of a DNA/RNA heteroduplex in the complex.

Base Sequence↗

PubMed1978

Gene enrichment using antibodies to DNA/RNA hybrids: purification and mapping of Dictyostelium discoideum rDNA.

Antibodies, shown to be specific for DNA/RNA hybrids, have been covalently attached to CNBr-activated Sepharose. The resultant affinity resin specifically binds DNA/RNA hybrids and has been used to enrich for the DNA which codes for rRNA in the slime mold Dictyostelium discoideum. By utilizing the technique of R-loop formation, DNA molecules containing the rRNA genes were isolated from total nuclear DNA in a double-stranded form. These rDNA molecules, which were recovered by high salt elution from the affinity resin, were typically 15-40 kbp in length, and thus contained DNA sequences adjacent to the selected sequences coding for the 17S and 26S rRNAs. In addition, evidence has been obtained concerning the structure of Dictyostelium rDNA which agrees with the finding (Taylor et al. (1977) ICN-UCLA Symp. Mol. Cell. Biol. 8, 309-313) that the rDNA molecules are not covalently attached to the chromosomes of this organism.

Chromatography, Affinity↗