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Results for “RNA, Complementary”

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PubMed1986

GABAergic regulation of enkephalin in rat striatum: alterations in Met5-enkephalin level, precursor content and preproenkephalin messenger RNA abundance.

The influence of chronic activation of gamma-aminobutyric acid (GABA) system on Met5-enkephalin (ME) biosynthesis was investigated in male rats. Activation of GABA system was achieved by raising the brain GABA concentration with aminooxyacetic acid (AOAA) or gabaculine which inhibits GABA-transaminase the enzyme responsible for the catabolism of GABA. After a regimen of repeated administration of AOAA (80 mg/kg/day for 8 days), the GABA concentration in the striatum could be maintained 2-fold greater than control value. AOAA decreased the ME levels in the striatum in a dose (20, 40 and 80 mg/kg)- and time (1-, 2-, 4- and 8-day treatment(s)-dependent fashion. Gabaculine also decreased the ME level in the striatum. Changes in ME level were not observed in other brain regions such as hypothalamus, hippocampus, frontal cortex and medulla/pons. In order to understand the mechanism involved in the decrease in ME level, the biosynthesis of ME was assessed. The preproenkephalin messenger RNA abundance was quantitated by RNA-complementary DNA hybridization technique; the total RNA was isolated, dot-blotted and hybridized with a nick-translated complementary DNA probe from rat brain. Administration of AOAA (80 mg/kg/day) for 8 days increased the preproenkephalin messenger RNA abundance whereas 1-, 2- or 4-day treatments did not alter the levels significantly. A similar trend of response was observed in the cryptic ME level which is indicative of the precursor content. The results suggest that chronic activation of the GABA system induces a sustained release of ME; in order to replenish the depletion, the biosynthesis of ME is augmented.(ABSTRACT TRUNCATED AT 250 WORDS)

Aminooxyacetic Acid↗

PubMed2000

Synthesis and hybridization properties of polyamide based nucleic acid analogues incorporating pyrrolidine-derived nucleoamino acids.

Ndelta-Fmoc protected nucleoamino acids of type I (Base = T, C, A) have been synthesized and employed as building blocks for the construction of novel polyamide based nucleic acid analogues. Homopyrimidine oligomer A binds to complementary RNA with significant affinity and in a sequence-specific fashion, while no binding was observed to complementary DNA.

Amino Acids↗

PubMed2002

Synthesis and hybridization properties of RNA containing 8-chloroadenosine.

8-Chloroadenosine (8-Cl-Ado) has shown potential as a chemotherapeutic agent for the treatment of multiple myeloma and certain leukemias. 8-Cl-Ado treatment leads to a decrease in global RNA levels and incorporation of the analog into cellular RNA in malignant cells. To investigate the effects of 8-Cl-Ado modifications on RNA structure and function, an 8-Cl-Ado phosphoramidite and controlled-pore glass support were synthesized and used to introduce 8-Cl-Ado at internal and 3'- terminal positions, respectively. RNA oligonucleotides containing 8-chloroadenine (8-Cl-A) residues were synthesized and hybridized with complementary RNA strands. Circular dichroism spectroscopy of the resulting RNA duplexes revealed that the modified nucleobase does not perturb the overall A-form helix geometry. The thermal stabilities of 8-Cl-Ado modified duplexes were determined by UV thermal denaturation analysis and were compared with analogous natural duplexes containing standard and mismatched base pairs. The 8-Cl-Ado modification destabilizes RNA duplexes by approximately 5 kcal/mole, approximately as much as a U:U mismatched base pair. The duplex destabilization of 8-Cl-A may result from perturbation of Watson-Crick base pairing induced by conformational preferences of 8-halogenated nucleosides.

2-Chloroadenosine↗

PubMed1992

Inhibition of mouse hepatitis virus multiplication by an oligonucleotide complementary to the leader RNA.

An oligonucleotide complementary to a leader RNA of positive-stranded mouse hepatitis virus (MHV) was tested for the effect on the viral multiplication in mouse DBT cells. A 14-mer antisense oligonucleotide contained a sequence complementary to the conserved pentanucleotide sequence, UCUAA, of the leader RNA. A treatment of MHV-infected cells with the antisense oligonucleotide at concentrations from 5 to 25 microM had an inhibitory effect on the viral multiplication and reduced the synthesis of viral specific mRNA and proteins. No inhibitory effect was observed when the cells were treated with sense oligonucleotide and oligonucleotide which contained unrelated sequences at concentrations from 1 to 10 microM. These results showed that antisense oligonucleotide against the leader RNA reduced the multiplication of positive-stranded RNA virus, MHV.

Animals↗

PubMed1977

Identification of human DNA sequences complementary to chromosomal RNA with an improved RNA/DNA gradient hybridization technique.

Native DNA components in the human genome were identified with an improved RNA/DNA gradient hybridization technique. 3H-Labelled complementary RNA transcribed from total human DNA was hybridized to homologous DNA which had been mildly sheared to generate single-stranded ends. Unhybridized RNA was eliminated from the reaction mixture by column chromatography before the hybrids were subjected to CsCl gradient centrifugation. Nine radioactive peaks representing RNA/DNA hybrids were reproducibly found at buoyant densities of 1.687, 1.693, 1.696, 1.699, 1.702, 1.705, 1.708, 1.711 and 1.715 g/cm3. The technique was also utilized to hybridize I125-labelled human chromosomal RNA to DNA. Three distinct radioactive peaks were found at the heavy buoyant densities of 1.702, 1.705, and 1.708 g/cm3, suggesting a differential enrichment of DNA components with sequences complementary to chromosomal RNA.

Base Sequence↗

PubMed1982

Synthesis in vitro of full length genomic RNA and assembly of the nucleocapsid of vesicular stomatitis virus in a coupled transcription-translation system.

Synthesis of a small amount of 42S RNA in addition to the VSV specific mRNA species was observed in a coupled transcription-translation system containing ribonucleoprotein particles from L cell infected with vesicular stomatitis virus and nuclease-treated ribosomal extract obtained from uninfected HeLa cells. Analysis on a CsCl density gradient showed that the synthesized 42S RNA was associated with newly synthesized by protein as a nucleoprotein of bouyant density of 1.3 g/ml. The 42S RNA and the N protein present in the nucleoprotein were resistant to nuclease and protease, respectively. About 35% of the remaining 65% had a complementary polarity. The evidence presented here demonstrates that both the full length genomic and the complementary RNA are associated with N protein in the in vitro replication process. A template role for the complementary 42S RNA for replication of the genomic RNA is also suggested.

Animals↗

PubMed1993

Decreasing the level of translation initiation factor 4E with antisense RNA causes reversal of ras-mediated transformation and tumorigenesis of cloned rat embryo fibroblasts.

Transformation of cloned rat embryo fibroblasts (CREF) with the T24-ras oncogene results in loss of contact inhibition, growth in soft agar and tumor formation in nude mice. Previously we showed that in such cells (CREF T24), the phosphorylation rate of protein synthesis initiation factor 4E (eIF-4E) is increased, correlating with an increase in the general rate of protein synthesis. In the present study, we have expressed antisense RNA complementary to eIF-4E mRNA in CREF T24 cells using a stably integrated vector. Cells expressing antisense RNA (CREF T24/AS) contained 30-50% of the normal level of eIF-4E and exhibited many of the properties of untransformed cells. CREF T24 had a spindle-shaped, refractile appearance, whereas CREF T24/AS grew in ordered, parallel patterns and exhibited contact inhibition similar to untransformed CREF. The rates of growth and protein synthesis in CREF T24/AS were decreased compared to CREF T24 but were not as low as in CREF. The efficiency of growth in soft agar was 11-fold lower for CREF T24/AS compared with CREF T24. The latency period for tumor formation in nude mice was increased from 8 days for CREF T24 to 17-27 days for CREF T24/AS and various clonal lines derived from them. Cell lines established from these CREF T24/AS-derived tumors were shown to have partially regained the eIF-4E levels characteristic of CREF T24. These results demonstrate that many of the phenotypic alterations associated with ras-induced malignant transformation can be reversed by a moderate reduction of the translational initiation capacity and therefore may be mediated through a translational mechanism.

Animals↗

PubMed1999

A re-investigation of the ribonuclease sensitivity of a DNA demethylation reaction in chicken embryo and G8 mouse myoblasts.

Recently published results (Nucleic Acids Res. 26, 5573-5580, 1998) suggest that the ribonuclease sensitivity of the DNA demethylation reaction may be an experimental artifact due to the possible tight binding of the nucleases to the methylated DNA substrate. Using an improved protocol we show for two different systems that demethylation of hemimethylated DNA is indeed sensitive to micrococcal nuclease, requires RNA and is not an experimental artifact. The purified 5-MeC-DNA glycosylase from chicken embryos and G8 mouse myoblasts was first incubated for 5 min at 37 degrees C with micrococcal nuclease in the presence of Ca2+ in the absence of the DNA substrate. Upon blocking the nuclease activity by the addition of 25 mM EGTA, the DNA demethylation reaction was initiated by adding the labeled hemimethylated DNA substrate to the reaction mixture. Under these conditions the DNA demethylation reaction was abolished. In parallel controls, where the purified 5-MeC-DNA glycosylase was pre-incubated at 37 degrees C with the nuclease, Ca2+ and EGTA or with the nuclease and EGTA, RNA was not degraded and no inhibition of the demethylation reaction was obtained. As has already been shown for chicken embryos, the loss of 5-MeC-DNA glycosylase activity from G8 myoblasts following nuclease treatment can also be restored by the addition of synthetic RNA complementary to the methylated strand of the substrate DNA. No reactivation of 5-MeC-DNA glycosylase is obtained by complementation with a random RNA sequence, the RNA sequence complementary to the non-methylated strand or DNA, thus ruling out a non-specific competition of the RNA for the binding of the nuclease to the labeled DNA substrate.

Animals↗

PubMed2004

Salt-dependent heat capacity changes for RNA duplex formation.

Two short non-self-complementary RNA duplexes have been probed extensively to assess whether the heat capacity change (DeltaCP) for RNA duplex formation is sensitive to the solution ionic strength. Isothermal titration calorimetry data for duplex formation were collected as a function of temperature to obtain the DeltaCP values under each condition. Ionic strength was varied by repeating the measurements in the presence of 0.1-1.5 M added NaCl. In both cases, the DeltaCP for duplex formation was shown to have a significant dependence on the solution ionic strength, varying linearly with the log of the added NaCl. In each case, the DeltaCP became more negative at high ionic strength. Heat capacity changes have been much better studied in the area of protein folding where DeltaCP is not strongly dependent upon ionic strength but instead depends primarily on the burial of hydrophobic surface. Whereas hydrophobic burial is almost certainly involved in RNA duplex formation as well, the polyanionic nature of the backbone and the associated ion condensation effects are almost certainly a contributor to the observed ionic strength dependence of DeltaCPs for duplex formation. These data also hint at the possibility that nearest neighbor effects may be evident in the heat capacity changes associated with nucleic acid folding.

Base Sequence↗

PubMed1997

The ups and downs of nucleic acid duplex stability: structure-stability studies on chemically-modified DNA:RNA duplexes.

In an effort to discover novel oligonucleotide modifications for antisense therapeutics, we have prepared oligodeoxyribonucleotides containing more than 200 different modifications and measured their affinities for complementary RNA. These include modifications to the heterocyclic bases, the deoxy-ribose sugar and the phosphodiester linkage. From these results, we have been able to determine structure-activity relationships that correlate hybridization affinity with changes in oligonucleotide structure. Data for oligonucleotides containing modified pyrimidine nucleotides are presented. In general, modifications that resulted in the most stable duplexes contained a heteroatom at the 2'-position of the sugar. Other sugar modifications usually led to diminished hybrid stability. Most backbone modifications that led to improved hybridization restricted backbone mobility and resulted in an A-type sugar pucker for the residue 5'to the modified internucleotide linkage. Among the heterocycles, C-5-substituted pyrimidines stood out as substantially increasing duplex stability.

DNA↗

PubMed2005

Increased DNA microarray hybridization specificity using sscDNA targets.

BACKGROUND: The most widely used amplification method for microarray analysis of gene expression uses T7 RNA polymerase-driven in vitro transcription (IVT) to produce complementary RNA (cRNA) that can be hybridized to arrays. However, multiple rounds of amplification are required when assaying very small amounts of starting RNA. Moreover, certain cRNA-DNA mismatches are more stable than the analogous cDNA-DNA mismatches and this might increase non-specific hybridization. We sought to determine whether a recently developed linear isothermal amplification method (ribo-SPIA) that produces single stranded cDNA would offer advantages over traditional IVT-based methods for microarray-based analyses of transcript expression. RESULTS: A single round of ribo-SPIA amplification produced sufficient sscDNA for hybridizations when as little as 5 ng of starting total RNA was used. Comparisons of probe set signal intensities obtained from replicate amplifications showed consistently high correlations (r = 0.99). We compared gene expression in two different human RNA samples using ribo-SPIA. Compared with one round IVT, ribo-SPIA had a larger dynamic range and correlated better with quantitative PCR results even though we used 1000-fold less starting RNA. The improved dynamic range was associated with decreases in hybridization to mismatch control probes. CONCLUSION: The use of amplified sscDNA may offer substantial advantages over IVT-based amplification methods, especially when very limited amounts of starting RNA are available. The use of sscDNA targets instead of cRNA targets appears to improve hybridization specificity.

Animals↗

PubMed1978

The frequencies of transcription from the E- and L-strands of polyoma DNA.

Exhaustion type hybridization was used to measure the amount of nuclear virus RNA complementary to the early (E) and late (L) polyoma virus DNA strands. At 36 h after infection between 2.5 and 7.3% of the newly synthesized virus RNA was complementary to the E-strand (-strand) and between 92.7 and 97.5% was complementary to the L-strand (+strand). This proportion was independent of the labelling time, indicating similar accumulation of the E- and L-RNA transcripts in the nucleus. The nuclear E- and L-RNA transcripts sedimented in a similar manner through sucrose gradients.

Cell Line↗

PubMed2002

Antisense properties of tricyclo-DNA.

Tricyclo (tc)-DNA belongs to the class of conformationally constrained DNA analogs that show enhanced binding properties to DNA and RNA. We prepared tc-oligonucleotides up to 17 nt in length, and evaluated their binding efficiency and selectivity towards complementary RNA, their biological stability in serum, their RNase H inducing potential and their antisense activity in a cellular assay. Relative to RNA or 2'-O-Me-phosphorothioate (PS)-RNA, fully modified tc-oligodeoxynucleotides, 10-17 nt in length, show enhanced selectivity and enhanced thermal stability by approximately 1 degrees C/modification in binding to RNA targets. Tricyclodeoxyoligonucleotides are completely stable in heat-deactivated fetal calf serum at 37 degree C. Moreover, tc-DNA-RNA duplexes are not substrates for RNase H. To test for antisense effects in vivo, we used HeLa cell lines stably expressing the human beta-globin gene with two different point mutations in the second intron. These mutations lead to the inclusion of an aberrant exon in beta-globin mRNA. Lipofectamine-mediated delivery of a 17mer tc-oligodeoxynucleotide complementary to the 3'-cryptic splice site results in correction of aberrant splicing already at nanomolar concentrations with up to 100-fold enhanced efficiency relative to a 2'-O-Me-PS-RNA oligonucleotide of the same length and sequence. In contrast to 2'-O-Me-PS-RNA, tc-DNA shows antisense activity even in the absence of lipofectamine, albeit only at much higher oligonucleotide concentrations.

Alternative Splicing↗

PubMed1981

Mitochondrial transcription complex from Saccharomyces cerevisiae.

A DNA protein complex has been isolated from the mitochondria of Saccharomyces cerevisiae. The complex transcribes RNA complementary to mtDNA in a nonrandom manner. The RNA polymerase activity contained in the transcription complex is not dependent on the addition of exogenous template. The activity is rendered template-dependent by autolysis and can be further purified by heparin-Sepharose 4B chromatography. The activity is inhibited by heparin, Mn2+, and increasing ionic strength. The activity requires Mg2+ and ribonucleotides. The preferred template for the template dependent activity is poly[d(AT)]. The majority of the RNA synthesized by the transcription complex from endogenous DNA is complementary to the DNA strands directing the synthesis of the large and small ribosomal RNA. In yeast the 21 S and 14S rRNA genes are widely separated, therefore the transcription of these two regions but not of the intervening regions by the transcription complex suggests the existence of at least two transcriptional promoters on the yeast mitochondrial genome.

DNA, Fungal↗

PubMed2003

Efficient RNase H-directed cleavage of RNA promoted by antisense DNA or 2'F-ANA constructs containing acyclic nucleotide inserts.

The ability of modified antisense oligonucleotides (AONs) containing acyclic interresidue units to support RNase H-promoted cleavage of complementary RNA is described. Manipulation of the backbone and sugar geometries in these conformationally labile monomers shows great benefits in the enzymatic recognition of the nucleic acid hybrids, while highlighting the importance of local strand conformation on the hydrolytic efficiency of the enzyme more conclusively. Our results demonstrate that the duplexes support remarkably high levels of enzymatic degradation when treated with human RNase HII, making them efficient mimics of the native substrates. Furthermore, interesting linker-dependent modulation of enzymatic activity is observed during in vitro assays, suggesting a potential role for this AON class in an RNase H-dependent pathway of controlling RNA expression. Additionally, the butyl-modified 2'F-ANA AONs described in this work constitute the first examples of a nucleic acid species capable of eliciting high RNase H activity while possessing a highly flexible molecular architecture at predetermined sites along the AON.

Arabinose↗

PubMed1989

Interferon induction by viruses. XIX. Vesicular stomatitis virus--New Jersey: high multiplicity passages generate interferon-inducing, defective-interfering particles.

The infectious particles of plaque-derived, low multiplicity passaged wild-type VSV of New Jersey origin consistently induce about 1800 units of interferon (IFN)/10(7) aged chick embryo cells. This inducing capacity is sensitive to both uv radiation and heat (50 degrees). Virus obtained after two successive high multiplicity passages in GMK-Vero cells consistently induced over 25,000 units of IFN/10(7) cells. The IFN induction dose-response curve showed that one IFN-inducing particle (IFP) per cell sufficed to produce a quantum yield of IFN, but infection with two or more IFPs led initially to a marked suppression in the yield of IFN. IFN induction was attributed to two distinct defective particles that differed in size, both containing snap-back RNA, i.e., covalently linked, self-complementary [+/-]RNA. The IFN-inducing capacity of these defective-interfering particles was not inactivated by uv or heat. However heat did eliminate the IFN suppressing activity observed at higher multiplicities, implicating a heat-sensitive component in the virion as a regulator of IFN yield, and involving possibly the virion transcriptase and 3'-leader RNA product.

Animals↗

PubMed2003

Injection of exogenous RNA in amphibian oocytes leads to RNA level fluctuations which are sensitive to cordycepin, an RNA chain elongation terminator.

Using an in vivo heterologous system to study the stability of Xenopus laevis RNA injected into axolotl (Ambystoma mexicanum) fertilized eggs, we have previously observed unexpected fluctuations in RNA level during early development [Andéol et al., Differentiation 63 (1998) 69-79]. In this study, we further characterize this phenomenon and establish its existence during axolotl and Xenopus oogenesis, suggesting a phylogenetically conserved mechanism. The phenomenon can occur with a variety of exogenous sense and antisense substrates. RNase protection experiments establish that most of the molecules have the same polarity as the initially injected RNA. In addition, trace amount of complementary RNA (cRNA) can be detected the injected samples. Cordycepin prevent increases in RNA levels indicating the involvement of an RNA synthesis. These results indicate the existence of an in vivo post-transcriptional RNA amplification mechanism during the early development of amphibians.

Ambystoma mexicanum↗

PubMed1989

Rom transcript of plasmid ColE1.

The ColE1 Rom protein contributes to copy number control by affecting the rate of formation of a complex between RNA II, the precursor of the primer for DNA replication, and RNA I, a small RNA complementary to the 5' end of RNA II. Interaction of RNA I with RNA II can affect plasmid copy number by preventing primer formation. Although the RNA I and RNA II transcripts have been well characterized, the rom mRNA has not previously been detected. We have now identified the rom mRNA, and determined its start site both in vitro and in vivo. We also have found that the rom mRNA terminates at any of several sites, and that its synthesis is not autoregulated by the Rom protein. By using an internal standard RNA to estimate the efficiency of detection of the rom mRNA, its level was determined to be about 1 molecule per cell.

Bacterial Proteins↗