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PubMed1989

The isopropoxyacetic group for convenient base protection during solid-support synthesis of oligodeoxyribonucleotides and their triester analogs.

Isopropoxyacetic anhydride was successfully used for protection of exoaminofunctions of 2'-deoxyadenosine, -guanosine and -cytidine. N-isopropoxyacetylated nucleosides are stable under the conditions of the synthesis of oligodeoxyribonucleotides on the solid support. Removal of N-isopropoxyacetyl is much faster than that of commonly used benzoyl or isobutyryl groups viz. it is completed within the operation of cleavage of the oligodeoxyribonucleotide from the solid support. This observation enabled synthesis of -OCH2CH3 and -OCH2CF3 triesters, which hydrolyse partially or completely when standard deprotection conditions are applied.

Acetates↗

PubMed1992

The synthesis and properties of oligodeoxyribonucleotides containing N6-methoxyadenine.

Oligodeoxyribonucleotides containing N6-methoxyadenine (M) have been synthesized. The order of stability of duplexes consisting of synthesized oligodeoxyribonucleotides, 5'd(CCTGGTAXCAGGTCC)3'-5'd(GGACCTGNTACCAGG)3' (X = M, A, G. N = A, G, T, C), was M: A (Tm = 52 degrees C) greater than M: T (50 degrees C) greater than M: G (48 degrees C) greater than M: C (46 degrees C) observed by thermal denaturation in a buffer of 0.01 M Na cacodylate, and 0.1 M NaCl at pH 7.0. The Tms are within a range of 6 degrees of difference, which is smaller than those of Tms of the duplexes containing A:N pairs (11 degrees) and G:N pairs (11 degrees). DNA replication study on a template-primer system, 5'd(32p-CAGCTTTCGC)3' 3'd(GTCGAAAGCGMAGTCG)5', showed that TTP and dCTP were incorporated into DNA strands at a site opposite to M by Klenow DNA polymerase, but dATP and dGTP were not.

Adenine↗

PubMed1995

Polyamine effects on purine-purine-pyrimidine triple helix formation by phosphodiester and phosphorothioate oligodeoxyribonucleotides.

Utilization of oligodeoxyribonucleotides to inhibit specific gene transcription in vivo (antigene strategy) requires the efficient formation of triple helices under physiological conditions. However, pyrimidine-motif triplexes are not favored at physiological pH, and physiological concentrations of potassium cations hamper purine-motif triplex formation. Here we investigated the effects of polyamines on promoting triplex formation by G/T-rich oligodeoxyribonucleotides containing either phosphodiester or a diastereomeric mixture of phosphorothioate linkages. Compared with Mg2+, equimolar concentrations of polyamines greatly facilitated purine-motif triplex formation with the following order of effectiveness: spermine > spermidine > putrescine. At low polyamine concentrations, phosphorothioate oligonucleotides were better at triplex formation than the corresponding phosphodiester oligonucleotides. Kinetic studies indicated that polyamines facilitated triplex formation by increasing the rate of oligonucleotide-duplex DNA association. However, triplex accumulation with either oligonucleotide was still low under physiological conditions (140 mM K+, 10 mM Mg2+, 1 mM spermine). The inhibitory effects of K+ could be partially overcome with high concentrations of Mg2+ or spermine, with phosphodiester oligonucleotides being better able to form triplexes than phosphorothioates under these conditions.

Base Sequence↗

PubMed1996

Cleavage of oligodeoxyribonucleotides from controlled-pore glass supports and their rapid deprotection by gaseous amines.

A novel method for the deprotection of oligodeoxyribonucleotides has been developed. Gaseous amines such as ammonia or methylamine were employed under pressure to achieve mild and rapid deprotection conditions. For example, oligodeoxyribonucleotides having a (tert-butyl)phenoxyacetyl group for the protection of the exocyclic amino function of cytosine, adenine and guanine were released from controlled-pore glass supports and fully deprotected by ammonia or methylamine under gas phase conditions, at room temperature, within 35 or 2 min respectively.

Adenine↗

PubMed1996

Synthesis and hydrolysis of oligodeoxyribonucleotides containing 2-aminopurine.

A new method is reported for the synthesis of oligodeoxyribonucleotides containing 2-aminopurine residues at selected sites. This method involves protection of the 2-aminopurine ribonucleoside, reduction to the deoxyribonucleoside and standard preparation of the 5'-0- (4,4'-dimethoxytrityl)-3'-O-(2-cyanoethyl)-N,N- diisopropylphosphoramidite. The 2-aminopurine phosphoramidite prepared by this method couples with high efficiency and is stable under standard automated synthesis conditions. The presence and location of the 2-aminopurine residue is easily verified by treatment of the oligodeoxyribonucleotide with hot piperidine. The mechanism for selective hydrolysis of the 2-aminopurine residue in alkaline solution is predominantly direct cleave of the glycosidic bond.

2-Aminopurine↗

PubMed1996

Use of 1,2,4-dithiazolidine-3,5-dione (DtsNH) and 3-ethoxy-1,2,4-dithiazoline-5-one (EDITH) for synthesis of phosphorothioate-containing oligodeoxyribonucleotides.

Previous methods for the preparation of phosphorothioate-containing oligodeoxyribonucleotides rely on the reaction of phosphite triesters with sulfurizing reagents such as tetraethylthiuram disulfide (TETD) and 3H-1,2-benzodithiol-3-one 1,1-dioxide (Beaucage reagent). However, these and other sulfurizing reagents suffer from several disadvantages, and there is great impetus for the development of improved methods for sulfur transfer that are fully compatible with standard automated DNA synthesis. The present report describes the use of 1,2,4-dithiazolidine-3,5-dione (DtsNH) and 3-ethoxy-1,2,4-dithiazoline-5-one (EDITH) as effective sulfurizing reagents that meet these needs. Both reagents are easily prepared, and are stable upon prolonged room temperature storage in acetonitrile solution. The reagents are used at low concentrations (0.05 M) and for short reaction times (30 s). The methodology has been proven for the automated synthesis on 0.2-1.0 micromol scales of oligodeoxyribonucleotides, of length 6-20 bases, containing the phosphorothioate substitution at either a single site or at all positions.

Base Sequence↗

PubMed1997

Microwave-assisted rapid deprotection of oligodeoxyribonucleotides.

A novel method for the deprotection of oligodeoxyribonucleotides under microwave irradiation has been developed. The oligodeoxynucleotides having base labile, phenoxyacetyl (pac), protection for exocyclic amino functions were fully deprotected in 0. 2 M sodium hydroxide (methanol:water : : 1:1, v/v) = A and 1 M sodium hydroxide (methanol:water : : 1:1, v/v) = B using microwaves in 4 and 2 min, respectively. The deprotection of oligodeoxyribonucleotides carrying conventional protecting groups, dAbz, dCbzand dGpac, for exocyclic amino functions was achieved in 4 min in B without any side product formation. The deprotected oligonucleotides were compared with the oligomers deprotected using standard deprotection conditions (29% aq. ammonia, 16 h, 55 degrees C) with respect to their retention time on HPLC and biological activity.

Kinetics↗

PubMed1998

Exploratory studies on azole carboxamides as nucleobase analogs: thermal denaturation studies on oligodeoxyribonucleotide duplexes containing pyrrole-3-carboxamide.

In order to study base pairing properties of the amide group in DNA duplexes, a nucleoside analog, 1-(2'-deoxy-beta-D-ribofuranosyl)pyrrole-3-carboxamide, was synthesized by a new route from the ester, methyl 1-(2'-deoxy-3',5'-di-O-p -toluoyl-beta-D-erythro-pentofuranosyl)pyrrole-3-carboxylate, obtained from the coupling reaction between 1-chloro-2-deoxy-3,5-di-O -toluoyl-d-erythropentofuranose and methyl pyrrole-3-carboxylate by treatment with dimethylaluminum amide. 1-(2'-Deoxy-beta-D-ribofuranosyl)pyrrole-3-carboxamide was incorporated into a series of oligodeoxyribonucleotides by solid-phase phosphoramidite technology. The corresponding oligodeoxyribonucleotides with 3-nitropyrrole in the same position in the sequence were synthesized for UV comparison of helix-coil transitions. The thermal melting studies indicate that pyrrole-3-carboxamide, which could conceptually adopt either a dA-like or a dI-like hydrogen bond conformation, pairs with significantly higher affinity to T than to dC. Pyrrole-3-carboxamide further resembles dA in the relative order of its base pairing preferences (T >dG >dA >dC). Theoretical calculations on the model compound N-methylpyrrole-3-carboxamide using density functional theory show little difference in the preference for a syntau versus anti conformation about the bond from pyrrole C3 to the amide carbonyl. The amide groups in both the minimized antitau and syntau conformations are twisted out of the plane of the pyrrole ring by 6-14 degrees. This twist may be one source of destabilization when the amide group is placed in the helix. Another contribution to the difference in stability between the base pairs of pyrrole-3-carboxamide with T and pyrrole-3-carboxamide with C may be the presence of a hydrogen bond in the former involving an acidic proton (N3-H of T).

Azoles↗

PubMed1999

Rapid conditions for the cleavage of oligodeoxyribonucleotides from cis-diol-bearing universal polymer supports and their deprotection.

Two sets of deprotection conditions have been evolved for the deprotection of oligodeoxyribonucleotides and their cleavage from commercially available cis -diol group-bearing universal polymer supports. In the first case, oligodeoxyribonucleotides anchored on the universal support were subjected to one of the standard deprotection conditions followed by treatment with aqueous 0.5 M sodium chloride + 0.2 M sodium hydroxide solution for 30 min at room temperature. In the second case, oligonucleotides bound to the universal support were treated with methanolic sodium hydroxide solution under microwave radiation to obtain fully deprotected oligomers within 4 min. Under both conditions, the cleavage of oligonucleotides from the support and their deprotection occurred quantitatively without any side product formation. The cleaved oligonucleotides were found to be identical in all respects (retention time on HPLC and biological activity in PCR) to the corresponding standard oligo-nucleotides.

Chromatography, High Pressure Liquid↗

PubMed1978

Antibodies elicited by defined oligodeoxyribonucleotide sequences.

Antibodies to the oligodeoxyribonucleotides d(pT)3, d(pT)4, d(pT)6 and d(pA-A-T-T) were elicited in rabbits by immunization with electrostatic complexes of the respective haptens with methylated bovine serum albumin (MBSA). The antisera were assayed by complement fixation using denatured DNA's of various sources as antigens. The specificities of the antibodies were determined by estimating the inhibition of the complement fixation reaction by defined oligodeoxyribonucleotides. The antibodies were shown to be specific for the sequence of the oligode-oxyribonucleotides or parts of it.

Animals↗

PubMed1979

Defined transversion mutations at a specific position in DNA using synthetic oligodeoxyribonucleotides as mutagens.

The oligodeoxyribonucleotides, pCCCAGCCTCAA, which is complementary to nucleotides 5274--4284 of bacteriophage phi X174 viral DNA , and pCCCAGCCTAAA, which corresponds to the same sequence with a C leads to A change at the ninth nucleotide, were synthesized enzymatically. The second of these oligonucleotides was used as a primer for E. coli DNA polymerase I, from which the 5'-exonculease has been removed by proteolysis (Klenow enzyme), on wild-type phi X174 viral DNA template. After ligation, this yielded closed circular heteroduplex DNA with a G, A mismatch at nucleotide 5276. Transfection of E. coli spheroplasts with the heteroduplex DNA produced phage mutated at this nucleotide (G leads to T in the viral DNA) with high efficiency (13%). The mutant DNA, which corresponds to the gene B mutant am16, was reverted (T leads to G) by the wild type oligonucleotide with an efficiency of 19%. The nucleotide changes were established by sequence determination of the mutated viral DNA using the enzymatic terminator method. The production of specific transversion mutations, together with a previous demonstration of specific transition mutations (1), established that short enzymatically synthesized oligodeoxyribonucleotides can be used to induce any class of single nucleotide replacement with high efficiency and thus provide a powerful tool for specific genetic manipulations in circular genomes like that of phi X174.

Bacteriophage phi X 174↗

PubMed1980

The complete amino acid sequence of human fibroblast interferon as deduced using synthetic oligodeoxyribonucleotide primers of reverse transcriptase.

Using synthetic oligodeoxyribonucleotides to prime the transcription of interferon mRNA and cDNA, we recently determined the mRNA sequence coding for the 47 amino-terminal amino acids of mature human fibroblast interferon (1). From this sequence, we have now synthesised an oligodeoxyribonucleotide that is homologous with the mRNA sequence coding for amino acids 42-45 and used it as a primer to selectively transcribe an interferon cDNA template. The sequence of the newly synthesised DNA predicted the sequence of amino acids 48-109 in the interferon polypeptide. By repeating this process with one more primer, we have determined the complete amino acid sequence of mature human fibroblast interferon, a polypeptide of 166 amino acids.

Amino Acid Sequence↗

PubMed2003

Biochemical properties of phosphonoacetate and thiophosphonoacetate oligodeoxyribonucleotides.

Phosphorus-modified phosphonoacetate and thiophosphonoacetate oligodeoxyribonucleotides were chemically synthesized and their biochemical properties evaluated. Under physiological pH, these DNA analogs possess negative charge and form stable, complementary A-like DNA:RNA heteroduplexes when analyzed via circular dichroism spectroscopy. Phosphonoacetate and thiophosphonoacetate oligomers were found to stimulate RNase H activity and to be completely resistant to degradation by snake venom phosphodiesterase, DNase I and HeLa cell nuclear extract. Further research has demonstrated that neutral, esterified forms of these analogs can be taken up by cells. Phosphonoacetate and thiophosphonoacetate oligomers therefore represent a new class of oligodeoxyribonucleotide analogs having phosphorus- carbon bonds with considerable potential for use in biological research.

Chromatography, High Pressure Liquid↗

PubMed1986

Synthetic oligodeoxyribonucleotide probes to detect Kanagawa phenomenon-positive Vibrio parahaemolyticus.

Synthetic oligodeoxyribonucleotide probes were used in the colony hybridization test to examine the association between the Kanagawa phenomenon (KP) and the thermostable direct hemolysin gene (tdh) of Vibrio parahaemolyticus. Representative V. parahaemolyticus strains with a variety of KP reactions and 17 other Vibrio species were examined for homology with four synthetic oligodeoxyribonucleotide probes (19 to 21 bases long) representing different regions of the tdh structural gene. Under stringent conditions, two of the probes were capable of distinguishing KP-positive V. parahaemolyticus from KP-negative or KP weak-positive V. parahaemolyticus which possesses mutated tdh genes. Vibrio hollisae strains hybridized with all four probes under reduced stringency, suggesting that they have tdh-related genes which are homologous but not identical to the tdh gene in all the regions examined. The results suggest that the colony hybridization test with the synthetic oligonucleotide probes is more suitable for the definitive determination of KP-positive strains than the hybridization with the larger gene probe or immunological assays.

DNA, Bacterial↗

PubMed2005

Inhibitory effect of human telomerase antisense oligodeoxyribonucleotides on the growth of gastric cancer cell lines in variant tumor pathological subtype.

AIM: To investigate the inhibitory effect of specialized human telomerase antisense oligodeoxyribonucleotides on the growth of well (MKN-28), moderately (SGC-7901) and poorly (MKN-45) differentiated gastric cancer cell lines under specific conditions and its inhibition mechanism, and to observe the correlation between the growth inhibition ratio and the tumor pathologic subtype of gastric cancer cells. METHODS: Telomerase activity in three gastric cancer cell lines of variant tumor pathologic subtype was determined by modified TRAP assay before and after the specialized human telomerase antisense oligodeoxyribonucleotides were dealt with under specific conditions. Effect of antisense oligomer under specific conditions of the growth and viability of gastric cancer cell lines was explored by using trypan blue dye exclusion assay, and cell apoptosis was detected by cell morphology observation, flow cytometry and TUNEL assay. RESULTS: Telomerase activity was detected in well, moderately and poorly differentiated gastric cancer cell lines (the quantification expression of telomerase activity was 43.7TPG, 56.5TPG, 76.7TPG, respectively). Telomerase activity was controlled to 30.2TPG, 36.3TPG and 35.2TPG for MKN-28, SGC-7901 and MKN-45 cell lines respectively after treatment with human telomerase antisense oligomers at the concentration of 5 mumol/L, and was entirely inhibited at 10 mumol/L, against the template region of telomerase RNA component, whereas no inhibition effect was detected in missense oligomers (P<0.05). After treatment with antisense oligomers at different concentrations under specific conditions for 96 h, significant growth inhibition effects were found in MKN-45 and SGC-7901 gastric cancer cell lines (the inhibition ratio was 40.89% and 71.28%), but not in MKN-28 cell lines (15.86%). The ratio of inactive SGC-7901 cells increased according to the prolongation of treatment from 48 to 96 h. Missense oligomers could not lead to the same effect (P<0.05). Apoptosis of SGC-7901 and MKN-45 cells was detected not only by morphology and TUNEL assay but also by flow cytometry. The apoptotic rate reached 33.56% for SGC-7901 cells and 44.75% for MKN-45 cells. CONCLUSION: The viability and proliferation of gastric cancer cells can be inhibited by antisense telomerase oligomers. The growth inhibition of gastric cancer cells is correlated with concentration, time and sequence specialty of antisense oligomers. The inhibition mechanism of antisense human telomerase oligomers depends not only on the sequence specialty but also on the biological characteristics of gastric cancer cell lines.

Apoptosis↗

PubMed2000

[Cleavage of RNA in hybrid duplexes by ribonuclease H from E. coli. I. Substrate properties of complexes formed by RNA and tandem of short oligodeoxyribonucleotides].

We studied the E. coli RNase H cleavage of a 5'-labeled RNA fragment within two hybrid duplexes with identical sequences, one of which is formed by RNA and a 20-mer oligodeoxyribonucleotide (RNA/p20), whereas the second, by RNA and a tandem of short oligodeoxyribonucleotides (octanucleotide: (RNA/tandem). It was shown that RNA in the RNA/p20 complex is hydrolyzed from the 3'-end to yield consecutively the 17-, 14-, 11-, 8-, and 5-mer 5'-labeled fragments. On hydrolysis of RNA in complex RNA/tandem, the same products were registered, but their accumulation rates in this case differed. Thus, the initial rates of accumulation of the 17- and 8-mer were close. Moreover, the accumulation of the final 5-mer differed considerably: in the RNA/tandem complex it appeared within first minutes of the reaction, but only after a considerable lag period in complex RNA/p20. These data testify that the tandem is involved not only in the consecutive accumulation of the shortened products (which is characteristic of complexes including extended oligonucleotides) but also in the parallel accumulation. This results from hydrolysis of each duplex segment formed by RNA and the short oligonucleotide of the tandem. Although the order of recognition and cleavage of RNA target by ribonuclease H depends on the type of the hybrid duplex, the destruction of RNA target within complex RNA/tandem and in complex with the full-size oligonucleotide occurs with a close effectiveness.

Electrophoresis, Polyacrylamide Gel↗

PubMed2000

[Bcl-2 antisense oligodeoxyribonucleotide increases apoptosis of lung carcinoma cells induced by cisplatin].

OBJECTIVE: To study the effect of antisense oligodeoxyribonucleotide (ODN) to apoptotic suppress gene bcl-2 on apoptosis of lung carcinoma cells induced by cisplatin. METHODS: The lung carcinoma cells expressing bcl-2 were chosen to participate in this experiment. Cultured cells were divided into 7 groups: ODN, nonsense, ODN + cisplatin, nonsense + cisplatin, cisplatin, lipofectin and control. Bcl-2 antisense or nonsense mixed with lipofectin was added into above corresponding cultured cells. After cultured for 6 hours, cisplatin was added into corresponding groups. The cells were cultured again for 16 hours. And then, the cells were smeared on slides. Apoptotic cells were labeled with TdT-mediated dUTP nick end labeling (TUNEL) method on cell smears. Apoptotic index (AI) was counted to show the percentage of apoptotic cancer cells. The immunocytochemistry was used to detect the expression of bcl-2 in carcinoma cells. RESULTS: The bcl-2 expression of cancer cell in ODN group was significantly decreased compared to the control and nonsense groups. The AI of ODN + cisplatin group was 16.4 +/- 1.7, cisplatin group 4.1 +/- 0.8, antisense group 5.9 +/- 0.2, nonsense group 3.3 +/- 0.7, nonsense + cisplatin 7.6 +/- 1.1, lipofectin 5.1 +/- 0.9, control group 3.6 +/- 0.6. The AI of antisense + cisplatin group was significantly higher than that of other groups. CONCLUSION: Antisense oligodeoxyribonucleotide to bcl-2 can inhibit significantly the expression of bcl-2 of lung cancer cells and increase apoptosis of cancer cells induced by cisplatin.

Antineoplastic Agents↗

PubMed2005

[Study on the modulation of estrogen receptor isoforms alpha in endometrial carcinoma cells using antisense oligodeoxyribonucleotides].

OBJECTIVE: To verify the roles of estrogen receptor alpha in the tumogenesis of endometrial cancer in relationship with estrogen and tamoxifen, and to explore an efficient way to modulate the expression of estrogen receptor (ER) isoform alpha to built up a model of endometrial cancer cells expressing predominant one isoform of ERs for ultimately probing therapeutical and preventive antiestrogen for endometrial cancer. METHODS: A series of oligodeoxyribonucleotides against regions of the human ERalpha was synthesized and tested in human endometrial cancer cell line (HEC-1B cells) that expressed functional ERalpha. The expressions of ER isoforms were detected by Western blot using specific antibody. The change of HEC-1B cell proliferation in response to 17beta-estradiol(E2) and tamoxifen(TAM) under the impact of antisense oligodeoxyribonucleotides (AS-ODN) was studied. RESULTS: (1) Transfection with AS-ODN directed against the human ERalpha significantly inhibited ERalpha protein expression. (2) The proliferation of endometrial cancer cell line HEC-1B could be increased by both E(2) and TAM. The cells lost the ability to proliferate in response to E(2) after being transfected with ERalpha-AS-ODN at hours 24, 48 and 72. After being transfected with ERalpha-AS-ODN, HEC-1B cells lost the ability to proliferate in response to TAM at hour 48. CONCLUSION: (1) AS-ODNs directed against the human ERalpha can inhibit the expression of ER alpha effectively. The method can be used to built up a model of endometrial cancer cells expressing predominant one isoform of ERs. (2) ERalpha may be the primary receptor in the proliferation of HEC-1B cells in response to 17beta-estradiol and tamoxifen.

Cell Proliferation↗