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PubMed1990

Circular dichroism studies of an oligodeoxyribonucleotide containing a hairpin loop made of a hexaethylene glycol chain: conformation and stability.

An oligodeoxyribonucleotide, d(GCTCACAAT-X-ATTGTGAGC), where X represents a hexaethylene glycol chain, was studied using circular dichroism spectroscopy. Its conformation and conformational stability were compared to those of compounds where X was replaced by four thymines and to the duplex of same sequence without loop. The compound with the hexaethylene glycol chain can form a hairpin looped structure as well as a bulged duplex structure. In both cases the duplex region of the oligodeoxyribonucleotide exhibits the same conformation. In similar conditions the oligodeoxyribonucleotide with a four thymines loop forms exclusively a hairpin structure. Comparison between the thermodynamic parameters (delta H, delta S, delta G) associated with the formation of the structure of the three compounds are presented. In the case of the compound with the hexaethylene glycol chain it is shown that the large increase in its melting temperature (by about 35 degrees C in our experimental conditions) when compared to the non looped structure is mainly due to the fact that its melting process is intramolecular (monomolecular) whereas the other one is bimolecular.

Base Sequence↗

PubMed1993

A novel method for site specific introduction of single model oxidative DNA lesions into oligodeoxyribonucleotides.

Calf thymus terminal deoxynucleotidyl transferase was used to incorporate several products of oxidative base damage onto the 3' end of oligodeoxyribonucleotides. Under the defined conditions described in this report, single residues of dihydrothymine, beta-ureidoisobutyric acid, thymine glycol, urea, 7-hydro-8-oxoadenine, 7-hydro-8-oxoguanine, 5-hydroxycytosine and 5-hydroxyuracil were incorporated into oligodeoxyribonucleotides of different lengths. The reaction is both efficient and cost effective. The 3' termini of the reaction products were suitable substrates for ligation by phage T4 DNA ligase, facilitating greatly the construction of oligodeoxyribonucleotides containing unique and site specific oxidative DNA lesions.

Base Sequence↗

PubMed1997

The synthesis and stability of oligodeoxyribonucleotides containing the deoxyadenosine mimic 1-(2'-deoxy-beta-D-ribofuranosyl)imidazole-4-carboxamide.

Oligodeoxyribonucleotides containing the nucleoside analog 1-(2'-deoxy-beta-D-ribofuranosyl) imidazole-4-carboxamide (1) were synthesized by solid phase phosphoramidite technology. Nucleoside 1, which contains a reactive exocyclic amide moiety, was incorporated into synthetic oligodeoxyribonucleotides with the use of a benzoyl protecting group. The corresponding oligodeoxyribonucleotides with dI or dA in the same position in the sequence were synthesized for UV comparison of helix-coil transitions. The thermal melting studies indicate that 1, which could conceptually adopt either a dA- or a dI-like hydrogen bond configuration, pairs with significantly higher affinity to T than to dC. Nucleoside 1 further resembles dA in the relative order of its base pairing preferences (T >dG >dA >dC), but may be less discriminating than dA in its bias for base pairing with T over dG.

Aminoimidazole Carboxamide↗

PubMed1998

A high throughput method to investigate oligodeoxyribonucleotide hybridization kinetics and thermodynamics.

We describe a high throughput microtiter-based assay to measure binding of oligodeoxyribonucleotides to nucleic acid targets. The assay utilizes oligodeoxyribonucleotide probes labeled with a highly chemiluminescent acridinium ester (AE). Reaction of AE with sodium sulfite renders it non-chemiluminescent. When an AE-labeled probe hybridizes to a target nucleic acid AE is protected from reaction with sodium sulfite and thus remains chemiluminescent. In contrast, unhybridized probe readily reacts with sodium sulfite and is rendered non-chemiluminescent. Hybridization of an AE-labeled probe to a target nucleic acid can therefore be detected without physical separation of unhybridized probe by treatment of the hybridization reaction with sodium sulfite and measurement of the remaining chemiluminescence. Using this method we measured hybridization rate constants and thermodynamic affinities of oligodeoxyribonucleotide probes binding to simple synthetic targets as well as large complex biological targets. The kinetic and thermodynamic parameters were measured with a high degree of accuracy and were in excellent agreement with values measured by other established techniques.

Base Sequence↗

PubMed2000

Covalent attachment of oligodeoxyribonucleotides to amine-modified Si (001) surfaces.

A recently described reaction for the UV-mediated attachment of alkenes to silicon surfaces is utilized as the basis for the preparation of functionalized silicon surfaces. UV light mediates the reaction of t-butyloxycarbonyl (t-BOC) protected omega-unsaturated aminoalkane (10-aminodec-1-ene) with hydrogen-terminated silicon (001). Removal of the t-BOC protecting group yields an aminodecane-modified silicon surface. The resultant amino groups can be coupled to thiol-modified oligodeoxyribonucleotides using a heterobifunctional crosslinker, permitting the preparation of DNA arrays. Two methods for controlling the surface density of oligodeoxyribonucleotides were explored: in the first, binary mixtures of 10-aminodec-1-ene and dodecene were utilized in the initial UV-mediated coupling reaction; a linear relationship was found between the mole fraction of aminodecene and the density of DNA hybridization sites. In the second, only a portion of the t-BOC protecting groups was removed from the surface by limiting the time allowed for the deprotection reaction. The oligodeoxyribonucleotide-modified surfaces were extremely stable and performed well in DNA hybridization assays. These surfaces provide an alternative to gold or glass for surface immobilization of oligonucleotides in DNA arrays as well as a route for the coupling of nucleic acid biomolecular recognition elements to semiconductor materials.

Alkanes↗

PubMedDate not supplied

[Cleavage of RNA in a hybrid duplex by E. coli ribonuclease H. III. Substrate characteristics of hybrid duplexes of RNA and oligodeoxyribonucleotides containing a bleomycin A5 residue].

The effect of the bleomycin A5 residue linked to four-, eight-, and twelve-mer oligodeoxyribonucleotides on the substrate properties of their tandem and continuous (with or without unmodified octanucleotide effectors) hybrid duplexes was studied using E. coli RNase H. The bleomycin derivatives of oligodeoxyribonucleotides were shown to form hybrid duplexes with practically the same thermostability as those formed by unmodified oligodeoxyribonucleotides. The RNA in the bleomycin-containing hybrid duplexes is cleaved by the E. coli RNase H; however, the initial hydrolysis rate (v0) is 2.6-3.4-fold reduced for the continuous duplexes. In the case of tandem hybrid complexes, the effect of bleomycin on v0 was less pronounced. We hypothesized that steric factors play a key role in the bleomycin inhibition and effectors probably determine the substrate properties of such hybrid complexes. Of all the tandem systems studied, the RNA duplex with the bleomycin-containing tetranucleotide flanked with two effectors displayed the best substrate properties. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2002, vol. 28, no. 4; see also http://www.maik.ru.

Bleomycin↗

PubMed1991

Inhibition of DNA chain elongation by a phosphotriester linkage in template oligodeoxyribonucleotides.

We synthesized two oligodeoxyribonucleotides bearing an isopropyl phosphotriester at defined positions using new phosphorobisamidite chemistry. Diastereomers were separated with HPLC and their template properties were analyzed. Priming oligodeoxyribonucleotides labeled with 32P at the 5'-end were annealed to the modified oligodeoxyribonucleotides and elongated with DNA polymerase I large fragment from Escherichia coli. Results show that the phosphotriester inhibits the DNA chain elongation partially and the extents of the inhibition are remarkably different between the two diastereomers.

Base Sequence↗

PubMed1986

Analysis and purification of synthetic large oligodeoxyribonucleotides by HPLC on RPC-5 like resin.

Large oligodeoxyribonucleotides (20-160 bases), synthesized by the phosphoramidite method, have been analyzed and purified by HPLC on a RPC-5 like resin (Neosorb LC). Linear gradient of NaClO4 solution containing 10 mM NaOH and 0.1 mM EDTA was carried out for the elution. Large oligodeoxyribonucleotides bearing 4,4'-dimethoxytrytyl (DMT) group were separated very well from the shorter failed by-products. After removal of the DMT group, the products were analyzed and purified by repeating HPLC on Neosorb LC. This HPLC system gave well resolution of the desired oligodeoxyribonucleotide (over 50 bases) from the base modified by-products with the same chain length. The chromatogram showed the presence of large amount of by-products in addition to the desired product when methylphosphoramidite method was employed for the DNA synthesis.

Chromatography, High Pressure Liquid↗

PubMed1997

In vivo production of oligodeoxyribonucleotides of specific sequences: application to antisense DNA.

Retrons, bacterial retroelements found in Gram-negative bacteria, are integrated into the bacterial genome expressing a reverse transcriptase related to eukaryotic reverse transcriptase. The bacterial reverse transcriptases are responsible for the production of multicopy, single-stranded (ms) DNA consisting of a short single-stranded DNA that is attached to an internal guanosine residue of an RNA molecule by a 2',5'-phosphodiester linkage. Reverse transcriptases use an RNA transcript from the retrons, not only as primer, but also as template for msDNA synthesis. By studying the structural requirement, it was found that for msDNA synthesis an internal region of msDNA can be replaced with other sequences. msDNA can thus be used as a vector for in vivo production of an oligodeoxyribonucleotide of a specific sequence. Artificial msDNAs containing a sequence complementary to part of the mRNA for the major outer membrane lipoprotein of Escherichia coli effectively inhibited lipoprotein biosynthesis upon induction of msDNA synthesis. This is the first demonstration of in vivo synthesis of oligodeoxyribonucleotides having antisense function. Since we have previously demonstrated that bacterial retrons are functional in eukaryotes producing msDNA in yeast and in mouse NIH/3T3 fibroblasts, the present system may also be used to produce a specific oligodeoxyribonucleotide inside the cells to regulate eukaryotic gene expression artificially. We also describe a method to produce cDNA to a specific cellular mRNA using the retron system.

Animals↗

PubMed1987

Role of nucleoside components and internucleotide phosphate groups of oligodeoxyribonucleotide template in its binding to human DNA polymerase alpha.

Affinity labelling of human placenta DNA polymerase alpha (EC 2.7.7.7) with the reactive oligodeoxyribonucleotide d(pT)2pC[Pt2+(NH3)2OH](pT)7 was used for quantitative analysis of enzyme interaction with oligodeoxyribonucleotides as templates. Dissociation constants and Gibb's energy values for different oligothymidylates d(pT)nT where n = 1-14 have been evaluated by competitive experiments of these ligands with Pt2+ reagent. The data obtained prove the formation of one Me2+-dependent electrostatic contact and a hydrogen bond between the enzyme and one phosphate of these templates. One may suppose that the hydrophobic interaction of any other monomeric link of oligodeoxyribonucleotides with the enzyme template site takes place.

DNA Polymerase II↗

PubMed1994

Properties of exonuclease-resistant, psoralen-conjugated oligodeoxyribonucleotides in vitro and in cell culture.

We have prepared oligodeoxyribonucleotides that are modified at the 3'-terminal with N4-(4-aminobutyl)deoxycytidine and derivatized at the 5'-end with a 4'-([N-(aminoethyl)amino]methyl)-4,5',8-trimethylpsoralen, (ae)AMT, and whose sequences are complementary to vesicular stomatitis virus (VSV), N-protein mRNA, (ae)AMT-II, or VSV M-protein mRNA, (ae)AMT-III. (ae)AMT-II cross-links exclusively to VSV N-mRNA when a mixture of the oligomer and poly(A+) RNA from VSV-infected cells is irradiated in vitro with long wavelength UV light at either 20 degrees or 37 degrees C. N4-(4-Aminobutyl)deoxycytidine at the 3'-end of (ae)AMT-II does not appear to affect the binding or cross-linking of the oligomer to its target RNA. Oligomer (ae)AMT-II is completely resistant to hydrolysis by the 3'-5'-exonuclease activity found in fetal calf serum whereas a similar oligomer, (ae)AMT-I, which contains a 3'-terminal deoxycytidine, is hydrolyzed within 30 min when incubated at 37 degrees C. Intact (ae)AMT-II was found in both the cell lysate and cell culture medium after 12 hr of incubation with mouse L-cells along with d-(ae)AMTpT, which appears to result from endonuclease degradation of the oligomer. In contrast no intact (ae)AMT-I was found in either the cell lysate or the culture medium after 1 hr incubation. Although 10 microM (ae)AMT-II had no effect on VSV-protein synthesis in either unirradiated or UV-irradiated VSV-infected mouse L-cells, 10 microM (ae)AMT-III inhibited VSV protein synthesis 30% in irradiated cells. These results show that introduction of a N4-(4-aminobutyl)deoxycytidine at the 3'-end of an oligodeoxyribonucleotide significantly increases the resistance of the oligomer to degradation by 3'-5'-exonucleases but does not interfere with its ability to bind selectively to complementary RNA. Further derivatization with psoralen creates an oligomer that can be triggered to cross-link with RNA in a sequence-specific manner, is taken up intact by mammalian cells in culture, and exhibits biological activity. In combination, these two modifications endow the oligodeoxyribonucleotide with novel properties that could be exploited in the design of antisense or antigene reagents for use in controlling gene expression in mammalian cells.

Animals↗

PubMed1998

Efficient synthesis of double dye-labeled oligodeoxyribonucleotide probes and their application in a real time PCR assay.

A fast cleaving non-nucleosidic tetramethylrhodamine dye-labeled support has been developed for automated synthesis of double dye-labeled oligodeoxyribonucleotides in high yield. A mixture (1:1:2) of t-butylamine:methanol:water is used for cleavage and deprotection of dye-labeled oligodeoxyribonucleotides without any degradation or modification of dyes and nucleobases. The cleavage rate of oligodeoxyribonucleotides is significantly increased by using a diglycolate ester linkage instead of the commonly used succinate linkage. These double dye-labeled probes are used in PCR for real time detection of a specific PCR product. Using a 5'-exonuclease assay, detected on the ABI PRISM 7700 Sequence Detection System, there was no distinguishable difference in performance of probes synthesized using the dye-labeled support compared with traditional post-synthetic attachment of rhodamine.

Base Sequence↗

PubMed2003

Oligodeoxyribonucleotide probe accessibility on a three-dimensional DNA microarray surface and the effect of hybridization time on the accuracy of expression ratios.

BACKGROUND: DNA microarrays are now routinely used to monitor the transcript levels of thousands of genes simultaneously. However, the array fabrication method, hybridization conditions, and oligodeoxyribonucleotide probe length can impact the performance of a DNA microarray platform. RESULTS: We demonstrate solution-phase hybridization behavior of probe:target interactions by showing a strong correlation between the effect of mismatches in probes attached to a three dimensional matrix of a microarray and solution-based, thermodynamic duplex melting studies. The effects of mismatches in the probes attached to the microarray also demonstrate that most, if not all, of the oligodeoxyribonucleotide is available for hybridization. Kinetic parameters were also investigated. As anticipated, hybridization signals increased in a transcript concentration-dependent manner, and mismatch specificity increased with hybridization time. Unexpectedly, hybridization time increased the accuracy of fold changes by relieving the compression observed in expression ratios, and this effect may be more dramatic for larger fold changes. CONCLUSIONS: Taken together, these studies demonstrate that a three-dimensional surface may enable use of shorter oligodeoxyribonucleotide probes and that hybridization time may be critical in improving the accuracy of microarray data.

DNA, Complementary↗

PubMed1994

Enzymatic amplification of synthetic oligodeoxyribonucleotides: implications for triplet repeat expansions in the human genome.

The triplet repeat sequences (CGG)n, (GCT)n, and (CAG)n, which naturally occur in the human genome, can be autonomously expanded in human DNA by an as yet unknown mechanism. These in part excessive expansions have been causally related to human genetic diseases, the fragile X (Martin-Bell) syndrome, to myotonic dystrophy (Curschmann-Steinert), to spinal and bulbar muscular atrophy (Kennedy disease), and recently to Huntington disease. A GCC trinucleotide repeat was found to be expanded and methylated in the fragile site FRAXE on the human X chromosome. These findings were associated with mental retardation (Knight et al., 1993). In spinocerebellar ataxia type 1 (SCA1), a polymorphic CAG repeat was found to be unstable and expanded in individuals with that disease (Orr et al., 1993). We have demonstrated in in vitro experiments that the synthetic oligodeoxyribonucleotides (CGG)17, (CGG)12, (GCC)17, (CG)25, (CTG)17, or (CAG)17 plus (GTC)17, in the absence of added natural DNA, can be expanded with Taq polymerase in the polymerase chain reaction (PCR). Some expansion can already be detected after 4 PCR cycles. The E. coli Klenow DNA polymerase also functions in a similar amplification and expansion reaction performed at 37 degrees C without cycling. Other oligodeoxyribonucleotides, like, (CGG)7, (CGGT)13, or (TAA)17, are devoid of this property or have very low activity. The cytidine-methylated polymers (GCC)17 or (CG)25 yield expansion products of considerably reduced chain lengths. The expansion of the polymer (CGG)17 is affected by cytidine methylation to a lesser degree. A specific sequence and/or secondary structure and high CG content appear to be requirements for this expansion reaction by a possible slippage mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

PubMed1983

Ion-exchange high-performance liquid chromatography of oligodeoxyribonucleotides using formamide.

The superiority of buffer systems containing formamide for the ion-exchange high-performance liquid chromatographic separation of oligodeoxyribonucleotide mixtures generated in solid-phase syntheses is illustrated. The resolutions achieved are compared to those achieved with the same mixtures in other eluting solvents. The use of formamide systems is recommended for oligodeoxyribonucleotide purification in general and is particularly valuable where the oligonucleotide of interest is highly self-complementary and/or rich in deoxyguanosine residues.

Chromatography, High Pressure Liquid↗

PubMed1987

High-performance liquid chromatographic analysis of oligodeoxyribonucleotide base composition.

A significantly improved method for base composition analysis of synthetic oligodeoxyribonucleotides is presented. This highly accurate and sensitive method used enzymatic digestion followed by high-resolution HPLC of the nucleosides to determine the empirical base composition of the parent compound. The enzymatic digestion reaction is quantitative and is not blocked by modified bases, thus allowing the degree of base deprotection and chemical modification to be assessed. Digestion data are presented for oligodeoxyribonucleotides which range from 18 to 150 bases in length with excellent agreement of experimental and theoretical composition. The method is also applicable to high-molecular-weight genomic DNA.

Alkaline Phosphatase↗

PubMed1991

Phosphorothioate analogues of oligodeoxyribonucleotide: synthesis and activity as inhibitors of replication of human immunodeficiency virus.

The modifications of oligodeoxyribonucleotides include replacement of the other chain either all-PS (S-ODNs), or end-capped with several PS (SO-ODNs) groups at both 3'- and 5'-ends. A general synthesis of phosphorothioate analogues of oligodeoxyribonucleotides is described using the new phosphite. In assays of HIV, oligomers (S-ODNs) with complete replacement of phosphodiesters with phosphorothioate groups were found to be very active. Finally of particular interest is S-ODNs-rev or tat (20mers) which possessed slightly higher anti-HIV activity than S-dC28 itself. By contrast, the unmodified oligomers (ODNs) as well as SO-ODNs did not have any inhibitory effect.

Antiviral Agents↗

PubMed1979

Site-specific mutagenesis using synthetic oligodeoxyribonucleotide primers: I. Optimum conditions and minimum ologodeoxyribonucleotide length.

A synthetic oligodeoxyribonucleotide mismatched at a single nucleotide to a specific complementary site on wild-type circular phi X174 DNA can be used to produce a defined point mutation after in vitro incorporation into closed circular duplex DNA by elongation with DNA polymerase and ligation followed by transfection of Escherichia coli (Hutchison et al., 1978; Gillam et al., 1979). The present study is an investigation of the optimum conditions required for the oligodeoxyribonucleotide-primed reaction for production of transition and transversion mutations in phi X174 DNA, using the large (Klenow) fragment of E. coli DNA polymerase I. Under optimum conditions up to 39% of the progeny of transfection are the desired mutant and significant mutation is observed using a heptadeoxyribonucleotide.

Bacteriophage phi X 174↗