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PubMed2005

Synthesis and properties of PNA oligomers containing orotic acid derivatives.

We have investigated the incorporation of C6-derivatives of uracil into polypyrimidine peptide nucleic acid oligomers (PNA). Starting with orotic acid (uracil-6-carboxylic acid) we have prepared a PNA monomer containing the methyl orotate nucleobase which is compatible with Fmoc-based synthesis. Treatment of the resin-bound oligomers with hydroxide or amines cleanly converted the ester to an orotic acid or orotamide-containing PNA. Alternatively, the methyl orotate-containing PNA was liberated from the resin by standard acidolysis. PNA bearing a modified nucleobase was found to hybridize to both poly(rA) and poly(dA). Complexes with poly(rA) were more stable than those with poly(dA) but both were destabilized relative to an unmodified PNA. Modification of a terminal residue was tolerated better than modification of an internal position. The type of charge provided by the modification affected the complex stability. In the worst case, an internal modification was nearly as detrimental as a base mismatch.

Base Composition↗
PubMed2004

Relationship between gene expression and GC-content in mammals: statistical significance and biological relevance.

Mammalian chromosomes are characterized by large-scale variations of DNA base composition (the so-called isochores). In contradiction with previous studies, Lercher et al. (Hum. Mol. Genet., 12, 2411, 2003) recently reported a strong correlation between gene expression breadth and GC-content, suggesting that there might be a selective pressure favoring the concentration of housekeeping genes in GC-rich isochores. We reassessed this issue by examining in human and mouse the correlation between gene expression and GC-content, using different measures of gene expression (EST, SAGE and microarray) and different measures of GC-content. We show that correlations between GC-content and expression are very weak, and may vary according to the method used to measure expression. Such weak correlations have a very low predictive value. The strong correlations reported by Lercher et al. (2003) are because of the fact that they measured variables over neighboring genes windows. We show here that using gene windows artificially enhances the correlation. The assertion that the expression of a given gene depends on the GC-content of the region where it is located is therefore not supported by the data.

Animals↗
PubMed1987

Inosine.adenine base pairs in a B-DNA duplex.

The structure of the synthetic deoxydodecamer d(C-G-C-I-A-A-T-T-A-G-C-G) has been determined by single crystal X-ray diffraction techniques at 2.5A resolution. The refinement converged with a crystallographic residual, R = 0.19 and the location of 64 solvent molecules. The sequence crystallises as a B-DNA helix with 10 Watson-Crick base-pairs (4 A.T. and 6 G.C) and 2 inosine.adenine (I.A) pairs. The present work shows that in the purine.purine base-pairs the adenine adopts syn orientation with respect to the furanose moiety while the inosine is in the trans (anti) orientation. Two hydrogen bonds link the I.A. base-pair, one between N-1(I) and N-7(A), the other between O-6(I) and N-6(A). This bulky purine.purine base-pair is incorporated in the double helix at two positions with little distortion of either local or global conformation. The pairing observed in this study is presented as a model for I.A base-pairs in RNA codon-anticodon interactions and may help explain the thermodynamic stability of inosine containing base-pairs. Conformational parameters and base stacking interactions are presented and where appropriate compared with those of the native compound, d(C-G-C-G-A-A-T-T-C-G-C-G) and with other studies of oligonucleotides containing purine.purine base-pairs.

Adenine↗
PubMed1989

Sequence specificity of the P6 pairing for splicing of the group I td intron of phage T4.

Seventeen non-directed td- (thymidylate synthase-deficient) splicing-defective mutations isolated in phage T4 were localized within the catalytic core of the ribozyme. All of the mutations occur in conserved structural elements that form part of the td intron core secondary structure. Remarkably, seven of the seventeen independently isolated mutations clustered in the dinucleotide 5' element (P6[5']) of the putative two-base-pair P6 stem. An analysis of this region was undertaken by site-directed mutagenesis of the plasmid-borne td gene, leading to the following findings: First, the short P6 pairing in the td secondary structure model was verified with appropriate P6[5'] and P6[3'] compensatory mutations. Second, all P6[5'] and P6[3'] mutants are defective in the first step of splicing, guanosine-dependent 5' splice site cleavage, whereas their activity at the 3' splice site is variable. Third, residual in vitro splicing activity of the mutants altered on only one side of the P6 pairing is correlated with the ability to form an alternative two-base-pair P6 stem. Fourth, the degree to which the compensatory mutants have their splicing activity restored is highly condition-dependent. Restoration of phenotype of the compensatory P6[5']:[3'] constructs is weak under stringent in vitro conditions as well as in vivo. This sequence specificity is consistent with phylogenetic conservation of the P6 pairing elements in group I introns, and suggests either structural constraints on the P6 stem or a dual function of one or both pairing elements.

Base Composition↗
PubMed1992

Increased specificity for antisense oligodeoxynucleotide targeting of RNA cleavage by RNase H using chimeric methylphosphonodiester/phosphodiester structures.

One of the inherent problems in the use of antisense oligodeoxynucleotides to ablate gene expression in cell cultures is that the stringency of hybridization in vivo is not subject to control and may be sub-optimal. Consequently, phosphodiester or phosphorothioate antisense effectors and non-targeted cellular RNA may form partial hybrids which are substrates for RNase H. Such processes could promote the sequence dependent inappropriate effects recently reported in the literature. We have attempted to resolve this problem by using chimeric methylphosphonodiester/phosphodiester oligodeoxynucleotides. In contrast to the extensive RNA degradation observed with all-phosphodiester oligodeoxynucleotides, highly modified chimeric antisense effectors displayed negligible, or undetectable, cleavage at non-target sites without significantly impaired activity at the target site. We also note that all of the all-phosphodiester oligodeoxynucleotides tested demonstrated inappropriate effects, and that such undesirable activity could vary widely between different sequences.

Amino Acid Sequence↗
PubMed1998

Parallel thermodynamic analysis of duplexes on oligodeoxyribonucleotide microchips.

A microchip method has been developed for massive and parallel thermodynamic analyses of DNA duplexes. Fluorescently labeled oligonucleotides were hybridized with oligonucleotides immobilized in the 100 x 100 x 20 mum gel pads of the microchips. The equilibrium melting curves for all microchip duplexes were measured in real time in parallel for all microchip duplexes. Thermodynamic data for perfect and mismatched duplexes that were obtained using the microchip method directly correlated with data obtained in solution. Fluorescent labels or longer linkers between the gel and the oligonucleotides appeared to have no significant effect on duplex stability. Extending the immobilized oligonucleotides with a four-base mixture from the 3'-end or one or two universal bases (5-nitroindole) from the 3'- and/or 5'-end increased the stabilities of their duplexes. These extensions were applied to increase the stabilities of the duplexes formed with short oligonucleotides in microchips, to significantly lessen the differences in melting curves of the AT- and GC-rich duplexes, and to improve discrimination of perfect duplexes from those containing poorly recognized terminal mismatches. This study explored a way to increase the efficiency of sequencing by hybridization on oligonucleotide microchips.

Acrylic Resins↗
PubMed2005

Enhanced RNA cleavage within bulge-loops by an artificial ribonuclease.

Cleavage of phosphodiester bonds by small ribonuclease mimics within different bulge-loops of RNA was investigated. Bulge-loops of different size (1-7 nt) and sequence composition were formed in a 3' terminal fragment of influenza virus M2 RNA (96 nt) by hybridization of complementary oligodeoxynucleotides. Small bulges (up to 4 nt) were readily formed upon oligonucleotide hybridization, whereas hybridization of the RNA to the oligonucleotides designed to produce larger bulges resulted in formation of several alternative structures. A synthetic ribonuclease mimic displaying Pyr-Pu cleavage specificity cleaved CpA motifs located within bulges faster than similar motifs within the rest of the RNA. In the presence of 10 mM MgCl2, 75% of the cleavage products resulted from the attack of this motif. Thus, selective RNA cleavage at a single target phosphodiester bond was achieved by using bulge forming oligonucleotides and a small ribonuclease A mimic.

Base Sequence↗
PubMed1995

A 6-kb upstream region of the human transthyretin gene can direct developmental, tissue-specific, and quantitatively normal expression in transgenic mouse.

To ascertain whether a 6-kb upstream region of the human transthyretin (TTR) gene contains the cis-element(s) required for proper specificity and level of expression, transgenic mice carrying the human mutant TTR gene containing either 6-kb (6.0-hMet30) or 0.6-kb (0.6-hMet30) of the upstream region were produced and studied. The 6.0-hMet30 gene was expressed in the yolk sac, liver, and choroid plexus, where the mouse endogenous TTR gene is also expressed. In contrast, expression of the 0.6-hMet30 gene was restricted to the yolk sac and liver. The expression levels of the 6.0-hMet30 gene in the liver and serum were similar to those of the mouse TTR gene, and about 10-fold those of the 0.6-hMet30 gene. Before birth, the developmental profiles of the expression of both transgenes in each tissue were similar to those of the mouse TTR gene. However, the expression levels of the 6.0-hMet30 gene in the liver and serum increased after birth to reach adult levels at an age of 4 weeks, while expression of the 0.6-hMet30 gene remained at a low level after birth. These results suggest that the 6-kb upstream sequence contains the cis-elements required for developmental, tissue-specific, and quantitatively normal expression.

Animals↗
PubMed1979

Netropsin . dG-dG-dA-dA-dT-dT-dC-dC complex. Antibiotic binding at adenine . thymine base pairs in the minor groove of the self-complementary octanucleotide duplex.

The structure of the netropsin . dG-dG-dA-dA-dT-dT-dC-dC complex (one antibiotic molecule/self-complementary octanucleodide duplex) and its dynamics as a function of temperature have been monitored by the nuclear magnetic resonances of the Watson-Crick protons, the nonexchangeable base and sugar protons and the backbone phosphates. The antibiotic forms a complex with the nucleic acid duplex at the dA . dT-containing tetranucleotide segment dA-dA-dT-dT, with slow migration amongst potential binding sites at low temperature. The downfield shifts in the exchangeable protons of netropsin on complex formation demonstrate the contributions of hydrogen-bonding interactions between the antibiotic and the nucleic acid to the stability of the complex. Complex formation results in changes in the glycosidic torsion angles of both thymidine residues and one deoxyadenosine residue as monitored by chemical shift changes in the thymine C-6 and adenine C-8 protons. The close proximity of the pyrrole rings of the antibiotic and the base-pair edges in the minor groove is manifested in the downfield shifts (0.3--0.5 ppm) of the pyrrole C-3 protons of netropsin and one adenine C-2 proton and one thymine N-3 base-pair proton on complex formation. The internucleotide phosphates of the octanucleotide undergo 31P chemical shift changes on addition of netropsin and these may reflect, in part, contributions from electrostatic interactions between the charged ends of the antibiotic and the backbone phosphates of the nucleic acid.

Base Composition↗
PubMed1984

The effect of single base-pair mismatches on the duplex stability of d(T-A-T-T-A-A-T-A-T-C-A-A-G-T-T-G) . d(C-A-A-C-T-T-G-A-T-A-T-T-A-A-T-A).

The stabilitye and dynamics of the duplex d(T-A-T-T-A-A--T-A-T-C-A-A-G-T-T-G) . d(C-A-A-C-T-T-G-A-T-A-T-T-A-A-T-A) has been studied by means of ultraviolet-melting, temperature-jump relaxation kinetics, stopped-flow and NMR spectroscopy. In addition, the influence of the mismatches A . A, G . T, A .C and T . C,-incorporated in this double helix through the introduction of non-complementary bases in the second strand, on these parameters has been investigated. The thermodynamic parameters characterizing the melting of the duplexes have been determined. Interestingly, a substantial decrease was observed for the values of the melting enthalpy when proceeding from 0.015 M to 0.1 M NaCl solutions. All duplexes that contain mismatches have melting temperatures below that of the totally complementary double helix. On the basis of NMR experiments and differences in the free enthalpy values between the totally complementary double helix and the duplexes with mismatches, it could be concluded that some degree of stacking of the two mispaired bases between the neighbouring base pairs is maintained. At 1 M NaCl the enthalpy and entropy of the helix-to-coil transition of the totally complementary double helix are in good agreement with values calculated on the basis of the thermodynamic data of Borer et al. [Borer, Ph. N., Dengler, B. & Tinoco, I. (1974) J. Mol. Biol. 86, 843--853] which were derived for RNA. The kinetics of the complementary duplex and duplexes with G . T and A . C mismatches were studied by means of stopped-flow and temperature-jump techniques. The rate constants of formation are the same for the three double helices. The decrease in stability of the duplexes with mismatches is therefore entirely due to an increase of the dissociation constant. In temperature-jump experiments very often a fast relaxation process is observed in addition to the relaxation characterizing the disruption of the double helix. This fast relaxation process is customarily attributed to base destacking in the single helix. By combining temperature-jump relaxation kinetics with NMR melting experiments, it is shown that at the low temperature side of the melting transition this fast relaxation process is caused by rapid changes in the double-helical structure.

Base Composition↗
PubMed1991

Analysis of the significance of two single-base-pair differences in the SL3-3 and Akv virus long terminal repeats.

Two single-base-pair differences between the long terminal repeats (LTRs) of the T-lymphomagenic murine retrovirus SL3-3 and nonleukemogenic Akv virus were tested for effects on activity of the LTRs. Evidence was obtained from electrophoretic mobility shift assays for the presence of at least one factor in both T and non-T cells that bound to the region of the viral enhancers that contained the differences. However, no significant differences in activity in expression assays were detected when the two base-pair differences were exchanged between the two LTRs. Therefore, they do not contribute to the higher activity of the SL3-3 LTR in T-lymphoma cell lines.

Animals↗
PubMedDate not supplied

[Detection of the DeltaF508 trinucleotide deletion in the human CFTR gene by UV immobilization on nylon of a complex of a PCR fragment with a highly specific probe].

Deletion delta F508 has been revealed in PCR-amplified regions of human gene CFTR by color detection of the hybridization complex obtained by ligation of a tandem of short oligonucleotides on a DNA template followed by UV immobilization on nylon. The method allows reliable detection of the three-nucleotide deletion (insertion). The nonspecific signal depends on the nucleotide composition of the biotinylated tandem component. A significant level of the specific signal was achieved by using the PCR-amplified DNA fragments of different length (200-400 bp) irrespective of the position of the tandem-binding site in their sequences.

Base Sequence↗
PubMed1980

Synthesis of the human insulin gene. Part IV. New synthetic deoxyribooligonucleotide adaptors and primer for DNA cloning and sequence analysis.

The chemical synthesis of four new deoxyribooligonucleotides to be used as adaptors in molecular cloning of DNA for expression studies is described. These are (i) start-adaptor, (ii) stop-adaptor, (iii) conversion adaptor to insert ribosomal binding site, and (iv) retrieving adaptor. We have also synthesized a 19-bases-long primer, 5'-TTGTAAAACGACGGCCAGT-3' to increase the speed of DNA sequence analysis and also demonstrated its application using single-stranded M13 DNa vector.

Base Composition↗
PubMed1996

A DNA-based method for rationally assembling nanoparticles into macroscopic materials.

Colloidal particles of metals and semiconductors have potentially useful optical, optoelectronic and material properties that derive from their small (nanoscopic) size. These properties might lead to applications including chemical sensors, spectroscopic enhancers, quantum dot and nanostructure fabrication, and microimaging methods. A great deal of control can now be exercised over the chemical composition, size and polydispersity of colloidal particles, and many methods have been developed for assembling them into useful aggregates and materials. Here we describe a method for assembling colloidal gold nanoparticles rationally and reversibly into macroscopic aggregates. The method involves attaching to the surfaces of two batches of 13-nm gold particles non-complementary DNA oligonucleotides capped with thiol groups, which bind to gold. When we add to the solution an oligonucleotide duplex with 'sticky ends' that are complementary to the two grafted sequences, the nanoparticles self-assemble into aggregates. This assembly process can be reversed by thermal denaturation. This strategy should now make it possible to tailor the optical, electronic and structural properties of the colloidal aggregates by using the specificity of DNA interactions to direct the interactions between particles of different size and composition.

Base Sequence↗
PubMed1996

TATA-box DNA binding activity and subunit composition for RNA polymerase III transcription factor IIIB from Xenopus laevis.

The RNA polymerase III transcription initiation factor TFIIIB contains the TATA-box-binding protein (TBP) and polymerase III-specific TBP-associated factors (TAFs). Previous studies have shown that DNA oligonucleotides containing the consensus TATA-box sequence inhibit polymerase III transcription, implying that the DNA binding domain of TBP is exposed in TFIIIB. We have investigated the TATA-box DNA binding activity of Xenopus TFIIIB, using transcription inhibition assays and a gel mobility shift assay. Gel shift competition assays with mutant and nonspecific DNAs demonstrate the specificity of the TFIIIB-TATA box DNA complex. The apparent dissociation constant for this protein-DNA interaction is approximately 0.4 nM, similar to the affinity of yeast TBP for the same sequence. TFIIIB transcriptional activity and TATA-box binding activity cofractionate during a series of four ion-exchange chromatographic steps, and reconstituted transcription reactions demonstrate that the TATA-box DNA-protein complex contains TFIIIB TAF activity. Polypeptides with apparent molecular masses of 75 and 92 kDa are associated with TBP in this complex. These polypeptides were renatured after elution from sodium dodecyl sulfate-gels and tested individually and in combination for TFIIIB TAF activity. Recombinant TBP along with protein fractions containing the 75- and 92-kDa polypeptides were sufficient to reconstitute TFIIIB transcriptional activity and DNA binding activity, suggesting that Xenopus TFIIIB is composed of TBP along with these polypeptides.

Animals↗
PubMed2004

Photoelectron spectroscopy of isolated multiply negatively charged oligonucleotides.

Ultraviolet photoelectron spectroscopy in an ion beam was used to investigate the electronic properties of isolated DNA oligonucleotides [dA(5)-4H](4-) and [dT(5)-4H](4-), carrying four excess negative charges. We find the fourth adiabatic electron affinity to be slightly negative for [dA(5)-4H](4-), while it is positive for [dT(5)-4H](4-). This implies a significant influence of the base composition on energetics, which is in turn relevant for analytic applications and also for charge transport properties.

Anions↗
PubMed2004

The solution synthesis of antisense oligonucleotide-peptide conjugates directly linked via phosphoramide bond by using a fragment coupling approach.

To improve antisense oligonucleotide penetration inside cells, conjugates of oligonucleotides and cell-penetrating peptides, covalently linked through a phosphoramide bond, were prepared by a fragment coupling approach in the liquid phase. Two methods were used for this synthesis, i.e., phosphorylation of a peptide amino group by an oligonucleotide terminal phosphate 1-hydroxybenzotriazole ester in aqueous media or condensation of phosphate and amino groups in presence of triphenylphosphine, 2,2'-dithiopyridine and 4-dimethylaminopyridine in organic media. Several oligonucleotides, including a 18-mer antisense oligodeoxyribonucleotide complementary to an internal coding region of the reporter gene of the green fluorescent protein (GFP) were prepared. Peptides derived from the third helix of the homeodomain of Antennapedia, the influenza envelope hemagglutinin subunit as well as melittin and polymyxin B were used for the conjugates' synthesis. The peptides with various amino acid composition were chosen to confirm that these coupling methods are of a general use.

Amides↗
PubMed1998

rRNA-targeted fluorescent in situ hybridization analysis of bacterial community structure in river water.

An improved in situ hybridization technique, HNPP-FISH, using 2-hydroxy-3-naphthoic acid 2'-phenylanilide phosphate (HNPP) and Fast Red TR was applied to analyse the community structure of planktonic bacteria in river water. Oligonucleotide probes specific for the domain Bacteria (EUB338) and five bacterial groups [Flavobacterium-Cytophaga; Burkholderia-Pseudomonas (rRNA III)-authentic Alcaligenes; Vibrio-Aeromonas; Pseudomonas (rRNA I): the genus Acinetobacter] were used to investigate the bacterial community structure at two sites differing in organic carbon pollution level. At the eutrophic site, 54-68% of all cells visualized by staining with DAPI (4',6-diamidino-2-phenylindole) could be detected with probe EUB338. In samples from the oligotrophic site, 39-45% of the total cells hybridized with EUB338. At the eutrophic site, approximately 50% of the total cells were identified with the five group-specific probes; the bacterial community structure was dominated by the Flavobacterium-Cytophaga group and Burkholderia-Pseudomonas (rRNA III)-authentic Alcaligenes group. At the oligotrophic site, only 26-38% of the total cells were identified with the five group-specific probes. The community structure at the oligotrophic site was similar to that at the eutrophic site, although the percentage of EUB338-detectable cells differed. No appreciable change was found in the community structure during the sampling period at either site. The improved HNPP-FISH technique should be a useful tool for the analysis of microbial community composition.

Bacteria↗