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At least 145 records · Page 8Linked to original sources
PubMed1994

Cyclic AMP inhibits phosphatidylinositol-coupled and -uncoupled mitogenic signals in T lymphocytes. Evidence that cAMP alters PKC-induced transcription regulation of members of the jun and fos family of genes.

T lymphocyte stimulation via the Ag receptor results in activation of phospholipase C gamma 1 that catalyses the hydrolysis of phosphatidylinositol (PI). The hydrolysis generates inositol phosphate and diacylglycerol, which in turn, increase intracellular Ca2+ concentration and activates protein kinase C, respectively. Agonists operating via the adenylate cyclase pathway or cell permeable cAMP analogues inhibit T cell activation by interfering with the PI-turnover. We have shown that dbcAMP inhibits PI-independent mitogenic signals in T cells after stimulation with TPA plus ionomycin. dbcAMP inhibited the TPA plus ionomycin-induced transcription of IL-2 and IL-2R genes in EL4 cells, suggesting interference with biochemic events downstream to PI hydrolysis and upstream to transcription of early activation genes. Because many of the early genes operating in T cell mitogenesis possess a TPA-response element (TRE) in their promoter region, we tested the effect of cAMP on the TRE-binding protein, TPA-response element (TRE) in their promoter region, we tested the effect of cAMP on the TRE-binding protein, AP-1. dbcAMP increased the binding activity of nuclear proteins consisting of Fos:Jun heterodimers to a TRE-containing oligonucleotide, but altered the composition of Jun proteins in the AP-1. Furthermore, the TPA plus ionomycin-induced transcription program of members of the jun and fos family of genes was altered by dbcAMP, suggesting that inhibition of T cell proliferation by dbcAMP is a consequence of intervention in transcriptional regulation by TRE-binding proteins.

Animals↗
PubMed1981

DNase A, a poly(dA) and poly(dT)-specific deoxyribonuclease from Achatina fulica. Further investigation on the specificity.

The degradation of heat-denatured and native calf thymus DNAs, poly(dA-dT), poly(dA-dC) . poly(dG-dT), and poly(dG-dC) by DNase A has been investigated with the main aim of providing background information for studying the specificity of the enzyme. The specificity of the DNase A was studied by determining the base compositions of 5'- and 3'-terminal nucleotides of oligonucleotides released by the enzyme. The 5'- and 3'-terminal nucleotide compositions were found to vary in the average chain length (Pn) range of 45 to 7 for degradations of heat-denatured calf thymus DNA at pH 4.0, 4.5, 5.0, and 5.5. When the heat-denatured DNA was digested at the optimal pH (pH 5.0), at the Pn = 45 dAdo was predominant (39%) and dThd was minor (9%) in 5'-terminals, whereas dThd was predominant (43%) and dAdo was minor (9%) in 3'-terminals. At Pn = 7, dAdo was even more predominant (49%) and there was very little dThd (7%) in 5'-terminals. No preference was seen in 3'-terminals. This finding indicates that change in the specificity takes place during digestion. The compositions of 5'- and 3'-terminal nucleotides of oligonucleotides released from poly(dA-dT) by exhaustive digestion of the enzyme showed pronounced preferences for dAdo in 5'-terminals (81%) and dThd in 3'-terminals (78%). The hexamer or above deoxyadenylate and the nonamer or above of thymidylate were good substrates of the enzyme. It can be concluded that DNase A is a novel cluster-specific DNase which recognizes and cleaves sequences of oligodeoxyadenylate and/or sequences of oligothymidylate.

Animals↗
PubMed2002

Ion-pair reversed-phase high-performance liquid chromatography analysis of oligonucleotides: retention prediction.

An ion-pair reversed-phase HPLC method was evaluated for the separation of synthetic oligonucleotides. Mass transfer in the stationary phase was found to be a major factor contributing to peak broadening on porous C18 stationary phases. A small sorbent particle size (2.5 microm), elevated temperature and a relatively slow flow-rate were utilized to enhance mass transfer. A short 50 mm column allows for an efficient separation up to 30mer oligonucleotides. The separation strategy consists of a shallow linear gradient of organic modifier, optimal initial gradient strength, and the use of an ion-pairing buffer. The triethylammonium acetate ion-pairing mobile phases have been traditionally used for oligonucleotide separations with good result. However, the oligonucleotide retention is affected by its nucleotide composition. We developed a mathematical model for the prediction of oligonucleotide retention from sequence and length. We used the model successfully to select the optimal initial gradient strength for fast HPLC purification of synthetic oligonucleotides. We also utilized ion-pairing mobile phases comprised of triethylamine (TEA) buffered by hexafluoroisopropanol (HFIP). The TEA-HFIP aqueous buffers are useful for a highly efficient and less sequence-dependent separation of heterooligonucleotides.

Base Sequence↗
PubMed1997

16S ribosomal RNA-targeted oligonucleotide probes for monitoring of intestinal tract bacteria.

BACKGROUND: The composition of a sample of faecal bacteria can be determined by culturing different dilutions on specific media. However, not all bacteria can be cultured and media are not always specific. With a culture-independent approach a more accurate picture of the composition of the intestinal flora may be obtained. METHODS: Fluorescently labelled oligonucleotide probes targeted at 16S ribosomal RNA sequences specific for a bacterial genus were designed and applied for fluorescence in situ hybridization (FISH) of bacteria in human faecal samples. RESULTS: The mean number of Bifidobacterium spp. and the total number of anaerobic bacteria per gram of faeces were determined by culturing and with the probe technique. Although in both cases the number of Bifidobacterium spp. was about the same, 2.38 x 10(9) and 2.45 x 10(9), it was found that the contribution of Bifidobacterium spp. to the total composition is overestimated due to the lower number of total anaerobic bacteria estimated by culturing. CONCLUSION: Genus-specific or group-specific fluorescent 16S rRNA probes may become an invaluable tool in gut ecology studies.

Bacteria, Anaerobic↗
PubMed2003

Composition of microbiota in content and mucus from cecae of broiler chickens as measured by fluorescent in situ hybridization with group-specific, 16S rRNA-targeted oligonucleotide probes.

Six group-specific 16S rRNA-targeted oligonucleotide probes were used to investigate the composition of the microbiota of cecal content and mucus from broiler chickens. Together, the probes hybridized to as many as 94.7% of the bacteria detectable with the universal probe Bact338 in the content of the cecum of 2-d-old chicks. Fewer bacteria gave signals with these probes as the birds aged, and coverage was as low as 76% for the bacteria in cecal content of a 6-wk-old chicken. In the cecal content of 2-d-old chicks, approximately 56, 34, and 3% of the bacteria detectable with the universal probe reacted with the probes Enter1432 (enterics), Lacto722 (Lactobacillus/Streptococcus/Enterococcus), and Bif164 (bifidobacteria), respectively. Probes Clept1240 (Clostridium leptum subgroup), Erec482 (Clostridium coccoides-Eubacterium rectale), and Bacto1080 (Bacteroides groups) did not produce signals. In cecal content from 1-wk-old chicks, all six probes gave signals, and in samples from 6-wk-old birds approximately 3, 9, 6, 32, 22, and 8% of the bacteria detectable with the universal probe hybridized with the probes Enter1432, Lacto722, Bif164, Clept1240, Erec482, and Bacto1080, respectively. At this age, the six probes detected the phylogenetic groups in similar proportions in the microbiota of cecal content and cecal mucus. The exception was the enterics probe because more bacteria from the mucus fraction than from cecal content gave signals with this probe (13.4 vs. 4.4%, P<0.001).

Aging↗
PubMedDate not supplied

Rapid routes of synthesis of chemically reactive and highly radioactively labeled alpha- and beta-oligonucleotide derivatives for in vivo studies.

Development of the antisense oligonucleotide strategy for the regulation of gene expression in vivo poses several problems: the stability of oligonucleotides toward intracellular nucleases, labeling of oligonucleotides with high specific radioactivity, improvements of penetration of oligonucleotides into living cells, and enhancement of antisense action by coupling of chemically active groups. In the present paper synthesis of highly radioactively labeled [32P]- and [35S]oligonucleotide derivatives is described starting from both natural (beta) and nuclease-resistant (alpha) anomers of oligonucleotides. Conditions for preparative phosphorylation and thiophosphorylation suitable for oligonucleotides of various lengths, base composition, and anomeric forms were established. The stability of the phosphoramide bond under in vivo experimental conditions was checked. The methods of terminal phosphate chemical activation and terminal thiophosphate alkylation were applied to synthesize oligonucleotides equipped with hydrophobic, intercalating, alkylating, and photoactivatable groups. In the case of porphyrin-oligonucleotide conjugates, a series of new monofunctional porphyrin derivatives bearing a free aliphatic amino group was developed.

Acridines↗
PubMed1997

[Sensitized photomodification of DNA with binary systems. II. Spectral photosensitivity. One- and two quantum sensitization].

The efficiency of the photomodification of target single-stranded DNA with a decanucleotide derivative of p-azidotetrafluorobenzamide (direct photomodification) and with its complexes with decanucleotide derivatives of pyrene complementary to the adjacent segment of the target (sensitized photomodification) was studied as a function of the wavelength of long-wave UV light. The sensitized photomodification occurs mainly by singlet-singlet energy transfer from pyrene to azide in their complementary complex, which allows a significant increase in the rate and level of photomodification. When irradiation occurred simultaneously in the UV and visible regions (365-580 nm), two-photon triplet-triplet sensitization was revealed for the first time, which leads to a still greater acceleration of the target modification and a change of its site-direction from the G11 to T13 residue. The change of the mode of sensitization depending on the irradiation conditions allows the regulation of the reactivity of the binary system of oligonucleotide derivatives without altering their composition.

Autoradiography↗
PubMed1976

Sequence determination of the 3' terminal T1 oligonucleotide of 18S ribosomal RNA.

We have reexamined the primary structure of the 3' terminal oligonucleotide of 18S RNA from chicken fibroblasts and have shown, contrary to previously published results that this extremity G-A-U-C-A-U-U-AOH is identical to that of the rabbit, drosophila and bombyx. Furthermore the electrophoretic mobility and composition of the 3' terminal oligonucleotides of 18S RNA from rat and human cells are similar to that of other RNAs and show that the identity of structure for this region of 18S RNA extends to include all tested species between yeast and man. This finding reveals a marked degree of evolutionary constraint on the structure of this region.

Animals↗
PubMed2005

[Quantitative analysis of cantide in plasma by capillary gel electrophoresis].

Cantide is a 20-mer antisense phosphorothioate oligonucleotide that inhibits telomerase catalytic subunit hTERT, pharmacologic results showed that it had promising antitumor activity. In order to study the pharmacokinetic properties of Cantide, a capillary gel electrophoretic (CGE) method with internal standard was used for the determination of Cantide in rat plasma. Cantide and the internal standard had approximately equal percentage of base composition. Extraction of the phosphorothioate oligonucleotides from plasma was accomplished using two solid-phase extraction columns, a strong anion-exchange column to remove plasma proteins and lipids, followed by a reversed-phase column to remove plasma salts. A second desalting step, achieved by dialysis utilizing a membrane, was required to remove residual ionic material from the extracted sample. The size of the capillary column was 31 cm x 100 microm i.d. with an effective length of 20 cm. The running buffer was a mixture of Tris-boric acid-urea (pH 8.5). The calibration curve was linear in the range of 12.5 - 400 mg/L, with correlation coefficient (r) of 0. 999 8. Intra-day and inter-day relative standard deviations (RSDs) for the extracted samples were 0.398% - 2.46% and 2.75% - 6.07%, respectively. The range of recoveries was 99.53% - 102.1%. The results demonstrate the high accuracy, stability and reproducibility of the procedure.

Animals↗
PubMed2004

Detection of G-quartet structure in a DNA aptamer stationary phase using a fluorescent dye.

The fluorescent porphyrin dye N-methylmesoporphyrin IX (NMM) was used to provide direct evidence of intramolecular G-quartet formation by an oligonucleotide immobilized at the inner surface of a fused silica capillary. The oligonucleotide is the thrombin-binding DNA aptamer, which has been used in several analytical applications, including a stationary phase for open tubular capillary electrochromatography. Spectroscopic studies of the dye in batch solutions of the aptamer and of an oligonucleotide with the same base composition, but in a different, "scrambled" sequence that does not form an intramolecular G-quartet, provided evidence of selective fluorescence enhancement of NMM by the aptamer in the intramolecular G-quartet structure. On-column experiments compared results for injections of NMM onto an aptamer-coated capillary, a capillary coated with the scrambled sequence oligonucleotide, and a bare fused silica capillary. Results show that while NMM adsorbs to both coated capillaries, the selective fluorescence enhancement provides evidence of the intramolecular G-quartet structure on the aptamer-coated capillary.

Base Sequence↗
PubMed1999

Antisense oligonucleotides to gastrin inhibit growth of human pancreatic cancer.

Human pancreatic cancer is stimulated by the autocrine production of gastrin. In this study, the effects of administration of antisense oligonucleotides to gastrin on growth of pancreatic cancer were evaluated in vitro and in vivo. Log phase BxPC-3 human pancreatic cancer cells in culture were exposed to increasing concentrations (0.5-10 microM) of a synthetic 20-mer antisense phosphorothioate oligonucleotide to gastrin for 48 h and growth was assessed by the cellular proliferation assay. Growth was inhibited up to 88% by anti-gastrin oligonucleotides in a dose-related fashion compared to cells treated with diluent or a randomized sequence with the same composition as the anti-gastrin oligonucleotide. In vivo nude mice bearing BxPC-3 xenografts were treated daily for 14 days with a 0.1-ml intratumoral injection of either anti-gastrin (5 microM), the scrambled sequence control phosphorothioate oligonucleotide (5 microM), or buffer. Tumors from the anti-gastrin-treated mice were significantly smaller in volume and weight and had less gastrin detected by radioimmunoassay than either controls. These results support the role of gastrin as a stimulatory peptide for growth of human pancreatic cancer. Antisense oligonucleotide to gastrin may have a role in the future treatment of patients with pancreatic cancer.

Animals↗
PubMed1983

Mechanistic study of E. coli DNA topoisomerase I: cleavage of oligonucleotides.

E. coli DNA topoisomerase I catalyzes DNA topoisomerization by transiently breaking and rejoining single DNA strands (1). When an enzyme-DNA incubation mixture is treated with alkaline or detergent, DNA strand cleavage occurs, and the enzyme becomes covalently linked to the 5'-phosphoryl end of the cleaved DNA (2). Using oligonucleotides of defined length and sequence composition, this cleavage reaction is utilized to study the mechanism of E. coli DNA topoisomerase I. dA7 is the shortest oligonucleotide tested that can be cleaved by the enzyme. dT8 is the shortest oligo(dT) that can be cleaved. The site of cleavage in both cases is four nucleotides from the 3' end of the oligonucleotide. No cleavage can be observed for oligo(dC) and oligo(dG) of length up to eleven bases long. dC15 and dC16 are cleaved at one tenth or less the efficiency of oligo(dA) and oligo(dT) of comparable length.

DNA Topoisomerases, Type I↗
PubMed1997

Inhibition of Rev·RRE complexation by triplex tethered oligonucleotide probes.

We have described a class of molecules, called tethered oligonucleotide probes (TOPs), that bind RNA on the basis of both sequence and structure. TOPs consist of two short oligonucleotides joined by a tether whose length and composition may be varied using chemical synthesis. In a triplex TOP, one oligonucleotide recognizes a short single-stranded region in a target RNA through the formation of Watson-Crick base pairs; the other oligonucleotide recognizes a short double-stranded region through the formation of Hoogsteen base pairs. Binding of triplex TOPs to an HIV-1 Rev Response Element RNA variant (RREAU) was measured by competition electrophoretic mobility shift analysis. Triplex TOP.RREAU stabilities ranged between -9.6 and -6.1 kcal mol-1 under physiological conditions of pH, salt, and temperature. Although the most stable triplex TOP.RREAU complex contained 12 contiguous U.AU triple helical base pairs, complexes containing only six or nine triple helical base pairs also formed. Triplex TOPs inhibited formation of the RRE.Rev complex with IC50 values that paralleled the dissociation constants of the analogous triplex TOP.RREAU complexes. In contrast to results obtained with TOPs that target two single-stranded RRE regions, inhibition of Rev.RREAU complexation by triplex TOPs did not require pre-incubation of RREAU and a TOP: triplex TOPs competed efficiently with Rev for RREAU and inhibited RREAU.Rev complexation at equilibrium.

Base Composition↗
PubMed2004

Selectivity of quadruplex DNA stationary phases toward amino acids in homodipeptides and alanyl dipeptides.

Series of dipeptides, including homodipeptides and alanyl dipeptides, were separated using quadruplex (G-quartet) DNA stationary phases in open-tubular capillary electrochromatography (OTCEC). The stationary phases were constructed by covalently attaching the DNA oligonucleotides to the inner capillary surface. Three different G-quartet forming oligonucleotides were investigated: the two-plane G-quartet forming thrombin-binding aptamer, the four-plane analogue of the thrombin-binding aptamer, and a two-plane oligonucleotide identical to the thrombin-binding aptamer except for the replacement of the guanine by thymine in the central bridging loop of the G-quartet structure. Results were compared with results obtained using capillary electrophoresis on a bare capillary and OTCEC using an oligonucleotide with the same base composition as the thrombin-binding aptamer but in a different sequence that does not allow G-quartet formation as the stationary phase.

Amino Acids↗
PubMed2000

Structural heterogeneity in intramolecular DNA triple helices.

Oligodeoxynucleotides designed to form intramolecular triple helices are widely used as model systems in thermodynamic and structural studies. We now report results from UV, Raman and NMR experiments demonstrating that the strand polarity, which also determines the orientation of the connecting loops, has a considerable impact on the formation and stability of pyr x pur x pyr triple helices. There are two types of monomolecular triplexes that can be defined by the location of their purine tract at either the 5'- or 3'-end of the sequence. We have examined four pairs of oligonucleotides with the same base composition but with reversed polarity that can fold into intramolecular triple helices with seven base triplets and two T4 loops under appropriate conditions. UV spectroscopic monitoring of thermal denaturation indicates a consistently higher thermal stability for the 5'-sequences at pH 5.0 in the absence of Mg2+ ions. Raman spectra provide evidence for the formation of triple helices at pH 5 for oligomers with purine tracts located at either the 5'- or 3'-end of the sequence. However, NMR measurements reveal considerable differences in the secondary structures formed by the two types of oligonucleotides. Thus, at acidic pH significant structural heterogeneity is observed for the 3'-sequences. Employing selectively 15N-labeled oligomers, NMR experiments indicate a folding pattern for the competing structures that at least partially changes both Hoogsteen and Watson-Crick base-base interactions.

Base Pairing↗
PubMed2005

Distance and affinity dependence of triplex-induced recombination.

Triplex-forming oligonucleotides (TFOs) have the potential to serve as gene therapeutic agents on the basis of their ability to mediate site-specific genome modification via induced recombination. However, high-affinity triplex formation is limited to polypurine/polypyrimidine sites in duplex DNA. Because of this sequence restriction, careful analysis is needed to identify suitable TFO target sites within or near genes of interest. We report here an examination of two key parameters which influence the efficiency of TFO-induced recombination: (1) binding affinity of the TFO for the target site and (2) the distance between the target site and the mutation to be corrected. To test the influence of binding affinity, we compared induced recombination in human cell-free extracts by a series of G-rich oligonucleotides with an identical base composition and an increasing number of mismatches in the third strand binding code. As the number of mismatches increased and, therefore, binding affinity decreased, induced recombination frequency also dropped. There was an apparent threshold at an equilibrium dissociation constant (K(d)) of 1 x 10(-)(7) M. In addition, TFO chemical modification with N,N-diethylethylenediamine (DEED) internucleoside linkages to confer improved binding was found to yield increased levels of induced recombination. To test the ability of triplex formation to induce recombination at a distance, episomal targets with informative reporter genes were constructed to contain polypurine TFO target sites at varying distances from the mutations to be corrected. TFO-induced recombination in mammalian cells between a plasmid vector and a donor oligonucleotide was detected at distances ranging from 24 to 750 bp. Together, these results indicate that TFO-induced recombination requires high-affinity binding but can affect sites hundreds of base pairs away from the position of triplex formation.

Animals↗
PubMed1997

Control of complexity constraints on combinatorial screening for preferred oligonucleotide hybridization sites on structured RNA.

We have explored the use of short (10-mer), fully sequence-randomized oligonucleotide libraries for affinity-based screening in solution for energetically preferred sites of hybridization of a model 47-nucleotide (nt) mutant Ha-ras mRNA stem-loop fragment. In characterizing the model, binding studies using either a gel mobility-shift assay or an RNase ONE footprinting assay indicated the presence of a greatly preferred hybridization site for individual antisense RNA oligonucleotides on the 5'-most side of the ras RNA 19-nt loop. However, initial attempts to affinity-titrate combinatorial uniform 2'-O-methyl-substituted oligonucleotide libraries for selective binding to this 5'-loop site using an RNase ONE footprinting assay that can discriminate between binding to different sites on ras RNA were unsuccessful. By reducing the complexity of the library to a mix of seven RNA oligonucleotides complementary to a range of sites on ras RNA and with no self-complements, footprinting evidence for binding was obtained but was characterized by ras RNA site-specific binding constants differing dramatically from binding constants for individual oligonucleotides. The library complexity was reduced further to three different cases of two RNA oligonucleotides, one of which for all cases was the highest affinity 5'-loop complement. Detailed kinetic and thermodynamic binding analyses revealed a good fit of the data to independent (5'-loop and ascending stem sites), competitive (overlapping 5'-loop sites), or mutually allosteric (5'-loop and 3'-loop sites) formalisms and an energetics description showed that ras 5'-loop site-specific binding could be achieved by affinity titration only for the independent case. Reconstruction of events with the full complexity library suggested that there was the emergence of multiple, linked binding interactions and implied that successful hybridization affinity screening would be achieved only if all possible bimolecular binding interactions of individual library oligonucleotides with target RNA could be made mutually independent. Accordingly, by holding the calculated concentration of unique oligonucleotide sequences of a full complexity DNA library well below the value for the dissociation constant for binding of individual complement to the 5'-loop site and then titrating the concentration of ras RNA through this value, hybridization specific to the 5'-side of the ras loop was demonstrated as assayed either by sequential gel mobility-shift resolution of bimolecular complexes and RNase ONE footprinting in situ in gel slices or by RNase H cleavage of complexes in solution. Because this strategy uses an unbiased oligonucleotide library it should combinatorially identify energetically preferred hybridization sites on folded RNA targets of any sequence and of undetermined structure. This should enable a focused in vitro optimization of antisense oligonucleotide length, sequence, and chemical composition for preferred site binding affinity and specificity which, in turn, may be expected to provide for enhanced biological potency and specificity (Lima et al., 1996). Finally, the complexity constraints encountered and the fundamental requirement to control them presented here also should be applicable to interactions with any biomolecule target of any chemical class of combinatorial library when screened in solution in pooled mixes.

Electrophoresis, Polyacrylamide Gel↗