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PubMed1997

Retroviral oligonucleotide distributions correlate with biased nucleotide compositions of retrovirus sequences, suggesting a duplicative stepwise molecular evolution.

A computer-assisted analysis was made of 24 complete nucleotide sequences selected from the vertebrate retroviruses to represent the ten viral groups. The conclusions of this analysis extend and strengthen the previously made hypothesis on the Moloney murine leukemia virus: The evolution of the nucleotide sequence appears to have occurred mainly through at least three overlapping levels of duplication: (1) The distributions of overrepresented (3-6)-mers are consistent with the universal rule of a trend toward TG/CT excess and with the persistence of a certain degree of symmetry between the two strands of DNA. This suggests one or several original tandemly repeated sequences and some inverted duplications. (2) The existence of two general core consensuses at the level of these (3-6)-mers supports the hypothesis of a common evolutionary origin of vertebrate retroviruses. Consensuses more specific to certain sequences are compatible with phylogenetic trees established independently. The consensuses could correspond to intermediary evolutionary stages. (3) Most of the (3-6)-mers with a significantly higher than average frequency appear to be internally repeated (with monomeric or oligomeric internal iterations) and seem to be at least partly the cause of the bias observed by other researchers at the level of retroviral nucleotide composition. They suggest a third evolutionary stage by slippage-like stepwise local duplications.

Animals↗
PubMed1976

Nucleotide clusters in deoxyribonucleic acids. XIII. Sequence analysis of the longer unique pyrimidine oligonucleotides of bacteriophage S13 DNA by a method using unlabeled atarting oligonucleotides.

A method has been designed for sequence analysis of unlabeled oligodeoxynucleotides of chain length up to 20 nucleotides with no restriction on base composition. The unlabeled oligonucleotide preparation, is partially degraded with spleen exonuclease to give a series of products each differing in size by one nucleotide. The oligonucleotides in the digest are 5'-32 P terminally labeled with [psi-32] P ATP and T4 polynucleotide kinase, the excess ATP removed by chromatography on Sephadex G-25 then the oligonucleotides fractionated according to change length on DEAE-Sephadex. Each isostich fraction is analyzed for base composition and the nucleotide at the 5' terminus determined by its 32P label, resulting in direct read off of the sequence up to the penultimate 3'- terminal nucleotide. The 3'-terminal dinucleotide is analyzed by DEAE-cellulose chromatography of the Sephadex G-25 dinucleotide fraction. The method has been demonstrated by sequence analysis of the unique longer pyrimidine oligonucleotides C5T6, C2T8, C6T4 and C6T3 from S13 DNA. The sequences have extensive internal sequence homology.

Base Sequence↗
PubMed2002

Synergistic activation of the human choline acetyltransferase gene by c-Myb and C/EBPbeta.

To elucidate regulatory mechanisms at the transcriptional level of the human choline acetyltransferase gene (hChAT) we performed cotransfections assays in NG108-15 and SN56 cells using ChAT-CAT reporter plasmids with c-Myb and C/EBPbeta expression plasmids. The hChAT gene has several promoters, one of which (promoter P2 or M-type) is both c-Myb and C/EBPbeta inducible as 3-4-fold trans-activation was obtained in both cell lines when using either c-Myb or C/EBPbeta expression vectors alone. The simultaneous expression of c-Myb and C/EBPbeta in the absence or presence of NGFI-C (egr4) leads respectively to a 15-fold and 32-fold synergistic transcriptional activation of promoter P2. In the region upstream of exon M (P2) we identified a functional composite element including a c-Myb next to a C/EBP binding site. An oligonucleotide containing the composite element confers c-Myb and C/EBPbeta responsiveness to a heterologous promoter which is reduced after mutation of the c-Myb binding site. We also show that the coactivators CBP/p300 are required for c-Myb and C/EBPbeta trans-activation function and that RARalpha, RXRalpha and T3R have an inhibitory action on the synergistic transcriptional activity of c-Myb and C/EBPbeta and propose a model to explain the phenomena. Taken together, the results suggest that the synergistic effect of c-Myb and C/EBPbeta, previously observed in the hematopoietic system, functions equally in the neuronal system.

Animals↗
PubMed1994

Influence of base composition on membrane binding and cellular uptake of 10-mer phosphorothioate oligonucleotides in Chinese hamster ovary (CHRC5) cells.

A key problem in antisense therapeutics is the relatively poor cell uptake of oligonucleotides and subsequent transport to the cytoplasm and nucleus. Although the chemical characteristics of oligonucleotides seem likely to affect their uptake by cells, little is known about this issue. In this article we explore the effect of base composition on oligonucleotide uptake. We show that phosphorothioate homo-G oligomers have a distinctly greater cellular uptake than other phosphorothioate homooligomers. This is probably due to a greater initial association with the plasma membrane, because homo-G oligomers show the greatest binding to liposome membranes, when tested at physiological ionic strength. Under different buffer conditions appreciable differences in membrane binding to liposomes were detected for the various homooligonucleotides.

Animals↗
PubMed1997

Identification of 2'-hydroxyl groups required for interaction of a tRNA anticodon stem-loop region with the ribosome.

Synthetic RNA stem loops corresponding to positions 28-42 in the anticodon region of tRNA(Phe) bind efficiently in an mRNA-dependent manner to ribosomes, whereas those made from DNA do not. In order to identify the positions where ribose is required, the anticodon stem-loop region of tRNA(Phe) (Escherichia coli) was synthesized chemically using a mixture of 2'-hydroxyl- and 2'-deoxynucleotide phosphoramidites. Oligonucleotides whose ribose composition allowed binding were retained selectively on nitrocellulose filters via binding to 30S ribosomal subunits. The binding-competent oligonucleotides were submitted to partial alkaline hydrolysis to identify the positions that were enriched for ribose. Quantification revealed a strong preference for a 2'-hydroxyl group at position U33. This was shown directly by the 50-fold lower binding affinity of a stem loop containing a single deoxyribose at position U33. Similarly, defective binding of the corresponding U33-2'-O-methyl-substituted stem-loop RNA suggests that absence of the 2'-hydroxyl group, rather than an altered sugar pucker, is responsible. Stem-loop oligoribonucleotides from different tRNAs with U33-deoxy substitutions showed similar, although quantitatively different effects, suggesting that intramolecular rather than tRNA-ribosome interactions are affected. Because the 2'-hydroxyl group of U33 was shown to be a major determinant of the U-turn of the anticodon loop in the crystal structure of tRNA(Phe) in yeast, our finding might indicate that the U-turn conformation in the anticodon loop is required and/or maintained when the tRNA is bound to the ribosomal P site.

Anticodon↗
PubMed2006

Electrochemical response of oligonucleotides on carbon paste electrode.

Electrochemical response of synthetic oligonucleotides with different DNA bases sequences was investigated to find relationships between a chain composition and a signal. All DNA mononucleotides present electroactivity at a carbon paste electrode yielding anodic peaks at potentials: 1.00 (GMP), 1.28 (AMP), 1.47 (TMP) and 1.53 V (CMP). Also 15-mer homooligonucleotides show respective anodic peaks. Electrochemical response of 15- and 19-mer oligonucleotides consisting of all four DNA bases in different amounts was determined by the composition of oligonucleotide chain. When the contribution of different bases in oligonucleotide was balanced two anodic peaks were obtained that can be attributed to guanine and adenine moieties. Thymine residue is shown as a separate peak in voltammogram when its content in oligonucleotide chain is close to 50% of the total number of bases. Cytosine also yields a peak at its significant contribution in oligonucleotide chain and both pyrimidinic moieties produce catalytic waves easier when one of them is dominating or when only one pyrimidine derivative is present in a chain. Guanine is the easiest oxidized base and it produces a peak even at its minimal contribution (one guanine residue in 19-mer oligonucleotide). Guanine peak potential is dependent on oligonucleotide concentration and oligonucleotide composition. The lowest oligonucleotide concentration detected by guanine peak was 12.5 nM whereas detected by thymine peak was 90 nM.

Base Sequence↗
PubMed2002

Phosphorothioate oligonucleotides, suramin and heparin inhibit DNA-dependent protein kinase activity.

Phosphorothioate oligonucleotides and suramin bind to heparin binding proteins including DNA polymerases, and inhibit their functions. In the present study, we report inhibition of DNA-dependent protein kinase activity by phosphorothioate oligonucleotides, suramin and heparin. Inhibitory effect of phosphorothioate oligonucleotides on DNA-dependent protein kinase activity was increased with length and reached a plateau at 36-mer. The base composition of phosphorothioate oligonucleotides did not affect the inhibitory effect. The inhibitory effect by phosphorothioate oligodeoxycytidine 36-mer can be about 200-fold greater than that by the phosphodiester oligodeoxycytidine 36-mer. The inhibitory effect was also observed with purified DNA-dependent protein kinase, which suggests direct interaction between DNA-dependent protein kinase and phosphorothioate oligonucleotides. DNA-dependent protein kinase will have different binding positions for double-stranded DNA and phosphorothioate oligodeoxycytidine 36-mer because they were not competitive in DNA-dependent protein kinase activation. Suramin and heparin inhibited DNA-dependent protein kinase activity with IC(50) of 1.7 microM and 0.27 microg ml(-1) respectively. DNA-dependent protein kinase activities and DNA double-stranded breaks repair in cultured cells were significantly suppressed by the treatment with suramin in vivo. Our present observations suggest that suramin may possibly result in sensitisation of cells to ionising radiation by inactivation of DNA-dependent protein kinase and the impairment of double-stranded breaks repair.

Antineoplastic Agents↗
PubMed1975

Comparative study of the 16S RNA's of Escherichia coli and Proteus vulgaris.

We have studied the primary structure of 16S ribosomal RNA from Proteus vulgaris. The oligonucleotides containing methylated bases appeared to be the same as those of Escherichia coli, with one exception. We have also studied the base composition of the oligonucleotides obtained after T1 ribonuclease digestion of 16S RNA. On the basis both of their position on the fingerprint and of their pancreatic ribonuclease analyses, approximately 25 appeared to differ from those found in the E. coli T1 fingerprints. From the isolation of large fragments arising from the action of endogeneous endonucleases, we have concluded that the RNA sequences of both species are very similar. We have shown that the 5' and 3' extremities of 16S RNA are mostly conserved. It appears that the regions which are known to interact with ribosomal proteins in E. coli (particularly S8 and S15) are also less modified. It is noteworthy that the sequence modifications which have been observed are clustered and often correspond to regions of heterogeneity in E. coli 16S RNA.

Alkaline Phosphatase↗
PubMed2026

Dual recognition drives site-directed G-quadruplex stabilization: Oligonucleotide design in G4 ligand-oligonucleotide conjugates.

G-quadruplex (G4) DNA structures are increasingly recognized for their roles in transcriptional regulation and genome stability, making them attractive therapeutic targets. Selective recognition of individual G4s remains challenging due to the high structural similarity among G4 motifs. G4 Ligand-Oligonucleotides conjugates (GL-Os) address this challenge by combining small-molecule G4 ligands with the sequence specificity of oligonucleotides, targeting sequences flanking the intended G4 target. Here, we systematically investigate how oligonucleotide length, backbone composition, and sequence complementarity govern GL-O binding, selectivity, and G4 stabilization. We show that effective G4 recognition depends on the interdependence between oligonucleotide hybridization and G4 ligand binding, such that both elements cooperatively reinforce complex stability and site specificity. Longer oligonucleotides promote more stable complexes and stronger G4 stabilization, whereas central mismatches disrupt this dual-recognition mechanism. Replacement of DNA with peptide nucleic acids (PNAs) enhances binding strength, thermal stability, and metabolic stability. Importantly, ligand conjugation redirects PNA oligonucleotides from nonspecific polymerase stalling toward selective G4 stabilization. Finally, we demonstrate receptor-mediated cellular uptake of modified GL-Os, supporting the feasibility of cellular delivery while highlighting remaining delivery barriers. Together, these findings show the molecular design principles governing GL-O behavior and provide a foundation for the future development and evaluation of selective G4-targeting therapeutics.

G-quadruplex DNA↗
PubMed1991

The effect of temperature and oligonucleotide primer length on the specificity and efficiency of amplification by the polymerase chain reaction.

The polymerase chain reaction (PCR) is most effectively performed using a thermostable DNA polymerase such as that isolated from Thermus aquaticus. Since temperature and oligonucleotide length are known to control the specificity of oligonucleotide hybridization, we have investigated the effect of oligonucleotide length, base composition, and the annealing temperature on the specificity and efficiency of amplification by the PCR. Generally, the specificity of PCR is controlled by the length of the oligonucleotide and/or the temperature of annealing of the primer to the template. An empirical relationship between oligonucleotide length and ability to support amplification was determined. This relationship allows for the design of specific oligonucleotide primers. A model is proposed which helps explain the observed dependence of PCR on annealing temperature and length of the primer.

Base Sequence↗
PubMed1996

Sequence composition effects on the stabilities of triple helix formation by oligonucleotides containing N7-deoxyguanosine.

A nonnatural nucleoside, 7-(2-deoxy-beta-D-erythro-pento-furanosyl)-guanine (d7G), mimics protonated cytosine and specifically binds GC base pairs within a pyrimidine - purine - pyrimidine triple helix. The differences in association constants (KT) determined by quantitative footprint titration experiments at neutral pH reveal dramatic sequence composition effects on the energetics of triple helix formation by oligonucleotides containing d7G. Purine tracts of sequence composition 5'-d(AAAAAGAGAGAGAGA)-3' are bound by oligonucleotide 5'-d(TTTTT7GT7GT7GT7GT7GT)-3' three orders of magnitude less strongly than by 5'-d(TTTTTmCTmCTmCTmCTmCT)-3' (KT = 1.5 x 10(6) M(-1) and KT > or = 3 x 10(9) M(-1) respectively). Conversely, purine tracts of sequence composition 5'-d(AAAAGAAAAGGGGGGA)-3' are bound by oligonucleotide 5'-d(TTTTmCTTTT7G7G7G7G7G7GT)-3' five orders of magnitude more strongly than by 5'-d(TTTTmCTTTTmCmCmCmCmCT)-3' (KT > or = 3 x 10(9) M(-1) and KT < 5 x 10(4) M(-1) respectively). The complementary nature of d7G and mC expands the repertoire of G-rich sequences which may be targeted by triple helix formation.

Base Composition↗
PubMed2001

Phosphoramidate oligonucleotides as potent antisense molecules in cells and in vivo.

Antisense oligonucleotides are designed to specifically hybridize to a target messenger RNA (mRNA) and interfere with the synthesis of the encoded protein. Uniformly modified oligonucleotides containing N3'-P5' phosphoramidate linkages exhibit (NP) extremely high-affinity binding to single-stranded RNA, do not induce RNase H activity, and are resistant to cellular nucleases. In the present work, we demonstrate that phosphoramidate oligonucleotides are effective at inhibiting gene expression at the mRNA level, by binding to their complementary target present in the 5'-untranslated region. Their mechanism of action was demonstrated by comparative analysis of three expression systems that differ only by the composition of the oligonucleotide target sequence (HIV-1 polypurine tract or PPT sequence) present just upstream from the AUG codon of the firefly luciferase reporter gene: the experiments have been done on isolated cells using oligonucleotide delivery mediated by cationic molecules or streptolysin O (SLO), and in vivo by oligonucleotide electrotransfer to skeletal muscle. In our experimental system phosphoramidate oligonucleotides act as potent and specific antisense agents by steric blocking of translation initiation; they may prove useful to modulate RNA metabolism while maintaining RNA integrity.

5' Untranslated Regions↗
PubMed1995

Relative thermodynamic stability of DNA, RNA, and DNA:RNA hybrid duplexes: relationship with base composition and structure.

Fourteen oligonucleotides 8-21 nucleotides in length and their complements were synthesized as DNA and RNA. For each sequence, four kinds of duplexes, DNA:DNA, RNA:RNA, DNA:RNA, and RNA:DNA, were prepared. Twelve sequences had A.T/U content varying from 25 to 80% and dPy content in the DNA strands varying from 0 to 100%. Thermodynamic stabilities of four duplexes for each sequence were determined in solution containing 100 mM Na+, 10 mM phosphate, and 0.1 mM EDTA, pH 7.1. CD spectra and electrophoretic mobility on native polyacrylamide gel were measured for most duplexes. Quantitative correlations of hybrid stability both with deoxypyrimidine content and, at fixed dPy content, with the fraction of A.T/U in duplexes were found. We also demonstrated that hybrids with 70-80% deoxypyrimidine DNA strand and a high or moderate A.T/U fraction displayed the highest relative stability compared to their RNA counterparts. Relationships of relative intensities of CD bands at 210 nm and relative electrophoretic mobilities of hybrids with relative hybrid stability suggested that hybrid conformation varies continuously between A- and B-form and is the decisive factor in relative hybrid stability.

Base Sequence↗
PubMed2003

Immobilization of DNA on glassy carbon electrodes for the development of affinity biosensors.

The adsorption and electrooxidation of nucleic acids on glassy carbon electrodes are evaluated by using chronopotentiometric stripping analysis. The influence of electrochemical pretreatments, supporting electrolyte, halides and monovalent cations levels as well as the role of the oligonucleotide length and composition, accumulation potential and time on the adsorption and further electrooxidation of oligo(dG)(11) and oligo(dG)(21) are discussed. The adsorption behavior of single and double stranded calf thymus DNA on untreated glassy carbon electrodes is also evaluated. Trace (microg/l) levels of the oligonucleotides and polynucleotides can be readily detected following short accumulation periods with detection limits of 25, 60, 126 and 219 microg/l for oligo(dG)(21), oligo(dG)(11), ss and ds calf thymus DNA, respectively. The confined DNA layers demonstrated to be stable in air, in 0.200 M acetate buffer pH 5.00 and in 0.020 M phosphate buffer pH 7.40+0.50 M NaCl.

Adsorption↗
PubMedDate not supplied

[Binding of unnatural alpha,beta-oligocytidylates with DNA duplexes].

Binding of short fluorescently labeled AT-containing DNA duplexes with modified oligocytidylates is studied. The latter are modified to contain unnatural alpha-anomers along with natural beta-nucleotides; the nucleotide composition is selected according to putative pattern of unconventional triplex formation between duplex and oligomer bases. Nondenaturing gel electrophoresis is used to study complexation of fluorescent duplexes with cytidyl oligomers and oligocytidylate self-association at low temperatures. A DNA duplex of random AT composition is shown to bind with an excess of the corresponding oligocytidylate in 0.1 M Tris-HCl in the presence of Mg2+. Binding is observed at neutral pH values, while more basic pH (8.0) prevents complexation of the AT duplex and oligocytidylate. Contrary to oligonucleotides of irregular composition, a regular dA30:dT30 duplex does not bind with the dC strand. It is also shown that alternating self-complementary duplex d(AT)16 and oligocytidylate d(CbetaCalpha)15 do not form complexes, and poly-dC self-associates are formed instead. The effect of 2'-O-methylation of the third strand on complex formation and self-association is also analyzed. The results suggest that a modified oligocytidylate binds with a random-composition duplex, albeit with lower efficiency.

Base Sequence↗
PubMed2002

Preferential hydrolysis of gap and bulge sites in DNA by Ce(IV)/EDTA complex.

A new strategy for site-selective DNA hydrolysis, which takes advantage of the difference in reactivity between the phosphodiester linkages at the target site and the others, is presented. As the molecular scissors, homogeneous Ce(IV)/ethylenediamine-N,N,N',N'-tetraacetate (EDTA) complex is used without being bound to any sequence-recognizing moiety. When a gap structure is formed at the target site by using two short oligonucleotides and the composite is treated with the Ce(IV)/EDTA complex at pH 7.0 and 37 degrees C, the gap site in the substrate DNA is preferentially hydrolyzed over the double-stranded portion of the DNA. Site-selective DNA scission is also achieved by forming a bulge structure at the target site with the use of the appropriate oligonucleotide. These site-selective scissions are based on the following two factors: (i) the phosphodiester linkages in a single-stranded DNA are far more susceptible to the hydrolysis by the Ce(IV) complex than are the linkages in double-stranded DNA, and (ii) the phosphodiester linkages in the bulge sites are still more reactive than those in single-stranded DNA. In both cases, the addition of spermine significantly accelerates the scission.

Base Sequence↗
PubMed1998

Identification and partial purification of human double strand RNase activity. A novel terminating mechanism for oligoribonucleotide antisense drugs.

We have identified a double strand RNase (dsRNase) activity that can serve as a novel mechanism for chimeric antisense oligonucleotides comprised of 2'-methoxy 5' and 3' "wings" on either side of an oligoribonucleotide gap. Antisense molecules targeted to the point mutation in codon 12 of Harvey Ras (Ha-Ras) mRNA resulted in a dose-dependent reduction in Ha-Ras RNA. Reduction in Ha-Ras RNA was dependent on the oligoribonucleotide gap size with the minimum gap size being four nucleotides. An antisense oligonucleotide of the same composition, but containing four mismatches, was inactive. When chimeric antisense oligonucleotides were prehybridized with 17-mer oligoribonucleotides, extracts prepared from T24 cells, cytosol, and nuclei resulted in cleavage in the oligoribonucleotide gap. Both strands were cleaved. Neither mammalian nor Escherichia coli RNase HI cleaved the duplex, nor did single strand nucleases. The dsRNase activity resulted in cleavage products with 5'-phosphate and 3'-hydroxyl termini. Partial purification of dsRNase from rat liver cytosolic and nuclear fractions was effected. The cytosolic enzyme was purified approximately 165-fold. It has an approximate molecular weight of 50,000-65,000, a pH optimum of approximately 7.0, requires divalent cations, and is inactivated by approximately 300 mM NaCl. It is inactivated by heat, proteinase K, and also by a number of detergents and several organic solvents.

Animals↗
PubMed2003

Spectroscopic studies of the interaction of ferrous bleomycin with DNA.

Bleomycin is an antibiotic used in cancer chemotherapy for its ability to achieve both single- and double-strand cleavage of DNA through abstraction of the deoxyribose C4'-H. Magnetic circular dichroism (MCD) and X-ray absorption (XAS) spectroscopies have been used to study the interaction of the biologically relevant FeIIBLM complex with DNA. Calf thymus DNA was used as the substrate as well as short oligonucleotides, including one with a preferred 5'-G-pyrimidine-3' cleavage site [d(GGAAGCTTCC)2] and one without [d(GGAAATTTCC)2]. DNA binding to FeIIBLM significantly perturbs the FeII active site, resulting in a change in intensity ratio of the d d transitions and a decrease in excited-state orbital splitting (5Eg). Although this effect is somewhat dependent on length and composition of the oligonucleotide, it is not correlated to the presence of a 5'-G-pyrimidine-3' cleavage site. No effect is observed on the charge-transfer transitions, indicating that the H-bonding recognition between the pyrimidine and guanine base does not perturb Fe-pyrimidine backbonding. Azide binding studies indicate that FeIIBLM bound to either oligomer has the same affinity for N3-. Parallel studies of BLM structural derivatives indicate that FeIIiso-PEPLM, in which the carbamoyl group is shifted on the mannose sugar, forms the same DNA-bound species as FeIIBLM. In contrast, FeIIDP-PEPLM, in which the -aminoalanine group is absent, forms a new species upon DNA binding. These data are consistent with a model in which the primary amine from the -aminoalanine is an FeII ligand and the mannose carbamoyl provides either a ligand to the FeII or significant second-sphere effects on the FeII site; intercalation of the bithiazole tail into the double helix likely brings the metal-bound complex close enough to the DNA to create steric interactions that remove the sugar groups from interaction with the FeII. The fact that the FeII active site is perturbed regardless of DNA sequence is consistent with the fact that cleavage is observed for both 5'-GC-3' and nonspecific oligomers and indicates that different reaction coordinates may be active, depending on orientation of the deoxyribose C4'-H.

Animals↗