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PubMed2006

Potent antiscrapie activities of degenerate phosphorothioate oligonucleotides.

Although transmissible spongiform encephalopathies (TSEs) are incurable, a key therapeutic approach is prevention of conversion of the normal, protease-sensitive form of prion protein (PrP-sen) to the disease-specific protease-resistant form of prion protein (PrP-res). Here degenerate phosphorothioate oligonucleotides (PS-ONs) are introduced as low-nM PrP-res conversion inhibitors with strong antiscrapie activities in vivo. Comparisons of various PS-ON analogs indicated that hydrophobicity and size were important, while base composition was only minimally influential. PS-ONs bound avidly to PrP-sen but could be displaced by sulfated glycan PrP-res inhibitors, indicating the presence of overlapping binding sites. Labeled PS-ONs also bound to PrP-sen on live cells and were internalized. This binding likely accounts for the antiscrapie activity. Prophylactic PS-ON treatments more than tripled scrapie survival periods in mice. Survival times also increased when PS-ONs were mixed with scrapie brain inoculum. With these antiscrapie activities and their much lower anticoagulant activities than that of pentosan polysulfate, degenerate PS-ONs are attractive new compounds for the treatment of TSEs.

Animals↗
PubMed1995

Determination of fast dynamics of nucleic acids by NMR.

Double-stranded oligonucleotides of < 10 base pairs are adequately described as an isotropic rotor, using the correlation time for the cytosine H6-H5 vector. For longer fragments, the cylindrical model should be used for detailed analysis of NOEs. The appropriate correlation times can be calculated using the formulae of Tirado and Garcia de la Torre or derived from measurements of the cross-relaxation rate constants for cytosine (or uridine) H6-H5. Order parameters describing the degree of motion of different vectors on the subnanosecond time scale vary substantially, with typical values of S2 > 0.8 for base vectors and 0.5-0.8 for intrasugar and base-sugar vectors. Order parameters for terminal nucleotides are typically significantly smaller than for internal nucleotides, which may also mean that their conformation will be less well determined in the formalism of a unique structure. The CSA relaxation rates of the phosphodiesters appear to be insensitive to internal motions and may, therefore, provide the most accurate estimate of the overall tumbling time in nucleic acid fragments. Using a combination of relaxation data for different nuclei and different spectrometer frequencies may be expected to yield detailed information about fast motions in nucleic acid fragments.

Anisotropy↗
PubMed1995

Sequence-specific purification of nucleic acids by PNA-controlled hybrid selection.

Using an oligohistidine peptide nucleic acids (oligohistidine-PNA) chimera, we have developed a rapid hybrid selection method that allows efficient, sequence-specific purification of a target nucleic acid. The method exploits two fundamental features of PNA. First, that PNA binds with high affinity and specificity to its complementary nucleic acid. Second, that amino acids are easily attached to the PNA oligomer during synthesis. We show that a (His)6-PNA chimera exhibits strong binding to chelated Ni2+ ions without compromising its native PNA hybridization properties. We further show that these characteristics allow the (His)6-PNA/DNA complex to be purified by the well-established method of metal ion affinity chromatography using a Ni(2+)-NTA (nitrilotriactic acid) resin. Specificity and efficiency are the touchstones of any nucleic acid purification scheme. We show that the specificity of the (His)6-PNA selection approach is such that oligonucleotides differing by only a single nucleotide can be selectively purified. We also show that large RNAs (2224 nucleotides) can be captured with high efficiency by using multiple (His)6-PNA probes. PNA can hybridize to nucleic acids in low-salt concentrations that destabilize native nucleic acid structures. We demonstrate that this property of PNA can be utilized to purify an oligonucleotide in which the target sequence forms part of an intramolecular stem/loop structure.

Base Composition↗
PubMed2004

Mean and variance of the Gibbs free energy of oligonucleotides in the nearest neighbor model under varying conditions.

MOTIVATION: In order to assess the stability of DNA-DNA hybridizations-for example during PCR primer design or oligonucleotide selection for microarrays-one needs to predict the change in Gibbs free energy DeltaG during hybridization. The nearest neighbor model provides a good compromise between accuracy and computational simplicity for this task. To determine optimal combinations of reaction parameters (temperature, salt concentration, oligonucleotide length and GC-content), one would like to understand how DeltaG depends on all of these parameters simultaneously. RESULTS: We derive analytic results about the distribution of nearest neighbor DeltaG values for a Bernoulli random sequence model (specified by oligonucleotide length and average GC-content) under given experimental conditions. We find that the distribution of DeltaG values is approximately Gaussian and provide exact formulas for expectation and variance.

Base Composition↗
PubMed2000

DnaB helicase stimulates primer synthesis activity on short oligonucleotide templates.

DnaB helicase stimulated the second-order RNA primer synthesis activity of primase by over 5000-fold on DNA templates that were 23 nucleotides long. This template length is the same as the DnaB helicase thermodynamic binding site size [Jezewska, M. J., and Bujalowski, W. (1996) Biochemistry 35, 2117-2128]. This phenomenal stimulation was achieved by increasing the template affinity of primase by over 300-fold and increasing the catalytic rate by over 15-fold. It was necessary to determine the optimal amount of DnaB helicase to achieve this stimulation because helicase stimulation was cooperative at low concentration and inhibitory at high helicase concentration. The cooperative stimulation at low concentration indicated the presence of a time-dependent assembly step that preceded the active state. Besides stimulating primase activity, DnaB helicase also prevented primase from synthesizing RNA primers that were longer than the template sequence. In the absence of DnaB helicase, the majority of primers synthesized by primase were longer than the template and were named "overlong primers" [Swart, J. R., and Griep, M. A. (1995) Biochemistry 34, 16097-16106]. In contrast, the helicase-stimulated RNA primers were from 10 to 14 nucleotides in length with the 12-mer representing the majority of the total RNA primers produced. It was shown that DnaB helicase stabilized the open or single-stranded conformation of the template, which favored the synthesis of the template-length-dependent primers. In contrast, when primase acted alone, it stabilized the 3'-end hairpin conformation of the template so that the template's 3'-hydroxyl served as a "DNA primer" from which primase elongated to create the overlong primers.

Adenosine Triphosphate↗
PubMed1993

A study of oligonucleotide reassociation using large arrays of oligonucleotides synthesised on a glass support.

An extensive analysis of oligonucleotide interactions was carried out by hybridising a synthetic pool of 256 10mers, A(C,T)8A, representing all oligopyrimidine octamer sequences to an array of four copies of all 256 different octapurine sequences. The resulting 256 duplexes were quantified by phosphorimaging and analysed to determine the dependence of duplex formation on base composition, sequence, and salt concentration. The results show that the base composition dependence of duplex formation can be reduced by high concentrations of tetramethylammonium chloride. This chaotropic solvent also increases duplex yield by up to fifty-fold.

Base Composition↗
PubMed1995

Subunit composition of G(o) proteins functionally coupling galanin receptors to voltage-gated calcium channels.

The neuropeptide galanin is widely expressed in the central nervous system and other tissues and induces different cellular reactions, e.g. hormone release from pituitary and inhibition of insulin release from pancreatic B cells. By microinjection of antisense oligonucleotides we studied the question as to which G proteins mediate the galanin-induced inhibition of voltage-gated Ca2+ channels in the rat pancreatic B-cell line RINm5F and in the rat pituitary cell line GH3. Injection of antisense oligonucleotides directed against alpha 01, beta 2, beta 3, gamma 2 and gamma 4 G protein subunits reduced the inhibition of Ca2+ channel current which was induced by galanin, whereas no change was seen after injection of cells with antisense oligonucleotides directed against alpha i, alpha q, alpha 11, alpha 14, alpha 15, beta 1, beta 4, gamma 1, gamma 3, gamma 5, or gamma 7 G protein subunits or with sense control oligonucleotides. In view of these data and of previous results, we conclude that the galanin receptors in GH3 and in RINm5F cells couple mainly to the G(0) protein consisting of alpha 01 beta 2 gamma 2 to inhibit Ca2+ channels and use alpha 01beta 3 gamma 4 less efficiently. The latter G protein composition was previously shown to be used by muscarinic M4 receptors to inhibit Ca2+ channels.

Amino Acid Sequence↗
PubMed1993

A Staphylococcus aureus-specific oligonucleotide probe derived from 16S rRNA gene sequences.

Genes encoding 16S rRNA were sequenced from 16 species of Staphylococcus. Sequence analysis highlighted a potential Staph. aureus-specific region and a complementary oligonucleotide probe was synthesized and its specificity tested. Northern blotting indicated molecular specificity, and dot blots to RNA from Staph. aureus, Staph. capitis, Staph. caprae, Staph. carnosus, Staph. caseolyticus, Staph. cohnii, Staph. epidermidis, Staph. gallinarum, Staph. haemolyticus, Staph. hominis, Staph. hyicus, Staph. saprophyticus, Staph. sciuri, Staph. simulans, Staph. warneri and Staph. xylosus indicated species-specificity.

Base Composition↗
PubMed1987

The human genome encodes at least three non-allellic G proteins with alpha i-type subunits.

The amino acid sequence and composition of alpha-subunits of signal transducing G proteins of the same kind appear to vary by no more than 2% from species to species. Here we isolated a human liver cDNA using an oligonucleotide complementary to the sequences encoding the pertussis toxin (PTX) ADP-ribosylation site of the alpha-subunit of the rat brain G protein called Gi. Its open reading frame characterizes it as an alpha i-type cDNA--as opposed to alpha o-type--but predicts an amino acid composition that differs by 7% and 14%, respectively, from two other human alpha i-type molecules. Together with human brain alpha i (type-1) and human monocyte alpha i (type-2), the new human liver alpha i cDNA (type-3) forms parts of a family of alpha i molecules. Type-3 alpha i cDNA hybridizes to a approximately 3.6 kilobase long mRNA and type-2 alpha i cDNA hybridizes to an mRNA species of approximately 2.7 kilobases. This indicates that the human genome has at least three non-allellic genes encoding non-alpha o-type PTX substrates and provides structural evidence for the hypothesis that distinct effector systems are regulated by similar but nevertheless distinct PTX substrates.

Amino Acid Sequence↗
PubMed1997

Activation volume of DNA duplex formation.

The denaturation-renaturation thermal hysteresis was used to investigate the kinetics of the helix-coil equilibrium of four 22-base pair homopurine-homopyrimidine duplex oligonucleotides with fractional G x C base pair content (f(G x C)) between 0.14 and 0.5. In 20 mM NaCl and 20 mM Tris-HCl at pH 7.0 and at hydrostatic pressures up to 200 MPa, a two-state bimolecular reaction mechanism adequately described the observed kinetics. At 1 MPa and 47 degrees C, the rate constant for helix formation, k1, increased by a factor of 210, and the reverse rate constant, k(-1), decreased by a factor of 420 upon increasing f(G x C) from 0.14 to 0.5. The activation energies for formation of the duplexes were negative and relatively insensitive to f(G x C). The pressure-induced change in the rate constants is related to the activation volume of the reaction step. Pressure causes k1 to become larger, and the magnitude of the change in k1 with pressure increases the lower the f(G x C) value. Thus, when f(G x C) = 0.14, the activation volume for forward reaction, delta V++(1), equals -20 mL/mol, while when f(G x C) = 0.5, delta V++(1) = -6.7 mL/mol. The rate constant for strand separation, k(-1), decreases at high pressure. The activation volume for this step, delta V++(1), varies from 17 to 1.6 mL/mol when f(G x C) = 0.14 and 0.5, respectively. The delta V for helix formation calculated from the activation parameters changed from -23 mL/mol when f(G x C) = 0.14 to -5.8 mL/mol when f(G x C) = 0.5. From extrapolation, it is estimated that the molar volume change for formation of G x C base pairs in homopurine-homopyrimidine sequences is approximately 0 mL/mol. Parameters calculated from kinetics of other two duplex molecules, when f(G x C) = 0.23 and 0.32, lie between these extremes.

Base Composition↗
PubMed1991

Synthesis and duplex stability of oligonucleotides containing adenine-guanine analogues.

The nucleosides N6-methoxy-2'-deoxyadenosine (dZ) and 2-amino-9-(2-deoxy-beta-ribofuranosyl)-6-methoxyaminopurine (dK) have been synthesised and converted into 5'-O-dimethoxytrityl 3'-(2-cyanoethyl N,N-diisopropylphosphoramidites). These monomers have been used in machine DNA synthesis to give a set of heptadecanucleotides containing up to three analogue nucleotides. The melting transitions (Tm) show that the 17-mer duplexes containing Z.T and Z.C base-pairs have closely similar stabilities, as have those containing K.T and K.C pairs. They are less stable than the corresponding fully complementary duplexes, but more stable than those containing mismatched pairs. This, in the case of dZ, is in accord with the amino-imino tautomeric ratio of approximately 1:4 observed for the nucleoside in methyl sulfoxide. The application of oligomers containing such "degenerate" bases in oligonucleotide probes and primers is discussed.

Adenine↗
PubMed1990

Molecular mechanics and dynamics of DNA-furocoumarin complexes: effect of the aromatization of the pyrone ring on the intercalation geometry.

Results of molecular mechanics and dynamics calculations on intercalation complexes of DNA with various furocoumarins (psoralen, angelicin, 7-methylpyrido[3,4-c]psoralen and 7-methylpyrido[4,3-c]psoralen) and their corresponding aromatized derivatives are presented. These calculations were undertaken with the aim to elucidate the roles of the pyrone and pyridine moieties in the interactions which tend to orient the furocoumarins and pyridopsoralens between DNA base pairs. It appears that the intercalation geometries are very similar for the furocoumarins and related aromatized compounds. Therefore the oxygen and nitrogen atoms of the pyrone and pyridine moieties are not important in the orientation of the drug within the oligonucleotide.

Base Composition↗
PubMed1999

Isolation of centrosomes from Spisula solidissima oocytes.

We have described methods for the preparation of lysates and isolation of centrosomes from parthenogenetically activated oocytes of the surf clam, S. solidissima. Although oocyte availability is seasonal, between June and August as much as 2 liters of lysate can be generated by a single person. Since lysate can be stored frozen at -80 degrees C with no apparent loss in centrosome-dependent microtubule nucleation, this is a convenient system for year-round experimentation. On average, per milliliter of frozen-stored lysate, 2 or 3 x 10(6) centrosomes can be obtained at 3000- or 4000-fold purification by sucrose-density gradient centrifugation. Centrosome fractions typically contain 6.0 x 10(-12) g of protein per centrosome (Vogel et al., 1997) and 140-200 micrograms of protein is usually obtained from a single run involving six sucrose-density gradients (12 ml of lysate). One person can easily run three preparations in a day, and thus 420-600 micrograms of centrosome protein could be prepared daily. Therefore, based on the effort of one individual, as much as 20-40 mg of centrosome protein could be prepared per year. Another convenient feature of the system is that once centrosomes are isolated, they can be stored in high sucrose media at -80 degrees C for years with little or no loss in microtubule nucleation potential. Once isolated, centrosomes can be used for protein analysis, ultrastructural studies, or in functional reconstitution assays (Vogel, 1997). In addition, these preparations offer the isolation of sufficient quantities of centrosome proteins to be used as antigens for generating centrosome-specific antibodies or for obtaining protein sequence for the purpose of antibody production or the design of oligonucleotide primers for isolating cDNA fragments coding for centrosome proteins. Thus, the preparations described offer a biochemical approach for defining centrosome composition. The methods described for immunofluorescence analysis of asters assembled in lysates offer rapid and convenient preparations for screening antibodies for centrosome localization and specificity. Finally, the ability to prepare large quantities of homogeneous centrosomes should enhance ultrastructural studies since many centrosomes can be sectioned and analyzed simultaneously by EM, avoiding the problem of having to hunt through sections of single cells to find a single centrosome for analysis. In addition, colloidal gold localization studies, using antibodies and EM to pinpoint the relative location of individual proteins, could be carried out on populations of centrosomes in the same preparation simultaneously, thus drastically expanding the quantity of data gathered. In conclusion, the clam oocyte system described here offers the potential for a combined structural and biochemical approach for identification of novel centrosome proteins and elucidation of the molecular basis of microtubule nucleation, centrosome assembly, and centrosome function.

Animals↗
PubMed1976

Nucleotide sequence study of mouse 5.8S ribosomal RNA.

The primary structure of 5.8S mouse ribosomal RNA has been studied and compared to the structures previously established for other animal species. The results obtained show that mouse 5.8S ribosomal RNA yields pancreatic oligonucleotides with the same nucleotide sequence as the homologous oligonucleotides from rat cells. Furthermore T1 oligonucleotides of 5.8S ribosomal RNA from rat, mouse and human cells behave identically on fingerprinting fractionation and have the same composition as judged by pancreatic digestion. These results strongly suggest that the primary structures of 5.8S ribosomal RNA from rat, mouse and human cells are identical. This identity of structure is also found when the presence of several modified bases (psi and methylated bases) is considered. The findings emphasize the remarkable evolutionary stability of ribosomal gene structure. Comparison of the terminal regional of 5.8S RNA with those of 18S RNA reveals differences which imply a more complex mechanism underlying the maturation of 45S precursor RNA than the finding of identical structure would have suggested.

Animals↗
PubMed1986

Studies on nucleic acid interactions. I. Stabilities of mini-duplexes (dG2A4XA4G2-dC2T4YT4C2) and self-complementary d(GGGAAXYTTCCC) containing deoxyinosine and other mismatched bases.

The thermal stability of DNA duplexes containing deoxyinosine in a pairing position in turn with each of the four major deoxynucleotides has been investigated by measuring ultraviolet-absorbance at different temperatures. d(G2A4 X A4G2) and d(C2T4YT4C2) were prepared by the solid-phase phosphotriester method. When X is deoxyinosine, the Tm values of the duplexes are in the order Y = dC greater than dA greater than dG greater than dT greater than dU. The Tm of other duplexes containing dG, dA and dT at X were also measured. Self-complementary duplexes d(GGGAAINTTCCC) showed the same order of stability with N being dC, dA, dG and dT. Thermal stabilities of duplexes containing dG instead of dI were compared with other matched and mismatched duplexes. The Tm values of sequence isomers containing purine-pyrimidine combinations were compared. Self-complementary duplexes containing G-C and A-T in the central positions showed Tm values ca. 10 degrees higher than those containing C-G and T-A in the same positions. Thermodynamic parameters and circular dichroism spectra of these oligonucleotides were compared.

Base Composition↗
PubMed1988

Thermodynamic studies of base pairing involving 2,6-diaminopurine.

The thermal stabilities of oligodeoxyribonucleotide duplexes containing 2,6-diaminopurine (D) matched with each of the four normal DNA bases were determined by optical melting techniques. Comparison of optical melting curves yielded relative stabilities for the D-containing standard base pairs in an otherwise identical base-pair sequence. The D:T pair was found to be more stable than the A:T pair in dC3DG3:dC3TG3, as stable as the A:T in dCT3DT3G:dCA3TA3G, and less stable than the A:T in dCA3DA3G:dCT7G. The order of stabilities for X:Y in the DNA duplex dCA3XA3G:dCT3YT3G is: (A:T) greater than (T:D) congruent to (D:T) greater than or equal to (T:A) greater than (C:D) congruent to (D:A) congruent to (D:G) greater than or equal to (D:C) congruent to (G:D) congruent to (D:D) greater than or equal to (A:D). Implications of these results for design of DNA oligonucleotide probes are discussed.

2-Aminopurine↗
PubMed2000

Analysis of intestinal flora development in breast-fed and formula-fed infants by using molecular identification and detection methods.

BACKGROUND: An obvious difference between breast-fed and formula-fed newborn infants is the development of the intestinal flora, considered to be of importance for protection against harmful micro-organisms and for the maturation of the intestinal immune system. In this study, novel molecular identification methods were used to verify the data obtained by traditional culture methods and to validate the culture independent fluorescent in situ hybridization (FISH) technique. METHODS: From each of six breast-fed and six formula-fed newborn infants, six fecal samples were obtained during the first 20 days of life. The microbial compositions of the samples were analyzed by culturing on specific media and by FISH, by using specific 16S rRNA-targeted oligonucleotide probes. The colonies growing on the media were identified by random amplified polymorphic DNA pattern analysis and by polymerase chain reaction amplification and subsequent analysis of the 16S rRNA gene. RESULTS: Molecular identification of the colonies showed that the selective media are insufficiently selective and unsuitable for quantitative analyses. Qualitative information from the culturing results combined with the data obtained by the FISH technique revealed initial colonization in all infants of a complex (adult-like) flora. After this initial colonization, a selection of bacterial strains began in all infants, in which Bifidobacterium strains played an important role. In all breast-fed infants, bifidobacteria become dominant, whereas in most formula-fed infants similar amounts of Bacteroides and bifidobacteria (approximately 40%) were found. The minor components of the fecal samples from breast-fed infants were mainly lactobacilli and streptococci; samples from formula-fed infants often contained staphylococci, Escherichia coli, and clostridia. CONCLUSIONS: This study confirms the differences in development of intestinal flora between breast-fed and formula-fed infants. The results obtained from the FISH technique were consistent. Although the repertoire of probes for this study was not yet complete, the FISH technique will probably become the method of reference for future studies designed to develop breast-fed-like intestinal flora in formula-fed infants.

Bacteroides↗
PubMed1981

Detection of proton-acceptor sites of hydrogen bonding in adenine X uracil base pairs by the use of 15N magnetic resonance.

2',3',5'-Tri-O-acetyl[1,3,7,9,amino-15N]adenosine(ac3Ado) and its 8-2H and 8-bromo derivatives (ac3[8-2H]Ado and ac3br8Ado) were synthesized from 95% 15N-enriched adenosine which was obtained by microbial fermentation. The chemical shifts and nuclear Overhauser effects of 15N NMR of the adenosine derivatives were measured by changing the concentration of the mixed 1-cyclohexyluracil (cHxUra) in chloroform. The limiting shift of each 15N resonance was calculated by using the association constants obtained from proton magnetic resonances of the ac3Ado/cHxUra and ac3br8Ado/cHxUra systems. From the quantitative analysis of the 15N chemical shifts of the N-1 and N-7 atoms it could be concluded that ac3Ado X cHxUra dimers prefer the Hoogsteen-type pair while the Watson-Crick-type pairs are predominant in the ac3br8Ado X cHxUra dimers. The N-3 atom, which does not locate at the interaction site, showed a fairly large induced shift. Thus the induced shift of 15N resonance by association does not always mean the involvement of the corresponding nucleus to the interaction site. The effect of intermolecular hydrogen bonding on the nuclear Overhauser enhancement of the 15N-7 resonance was observed in the ac3[8-2H]Ado/cHxUra mixture. The presence of the Hoogsteen-type hydrogen bonding was proposed by the use of 15N NMR spectra. The present information will be useful for the elucidation of non-Watson-Crick-type base-pair interaction in transfer TNA and oligonucleotides.

Adenine↗