PREPARATION OF OLIGODEOXYNUCLEOTIDE 5′-TRIPHOSPHATES USING SOLID SUPPORT APPROACH

We report a synthetic procedure for conversion of oligonucleotides to their 5′-triphosphate derivatives with moderate yield. The oligonucleotides were synthesized on solid support using standard phosphoramidite protocols. The DMT protection group was removed and the 5′-OH was phosphitylated using 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one followed by reaction with tributyammonium pyrophosphate and iodine oxidation. After subsequent removal from support and complete deprotection, the products were isolated by anion-exchange HPLC chromatography. Structures of several 5′-triphosphate derivatives have been proven by phosphorus NMR, Mass-spectrometry and by HPLC comparison with authentic samples.     

Polymer supported synthesis of aminooxyalkylated oligonucleotides,and some applications in the fabrication of microarrays

A new protocol has been described for solid phase preparation of 3′- and 5′-aminooxylalkylated oligonucleotides using commercially available reagents. This involves attachment of linker 4 either with an LCAA-CPG support via succinoylation followed by synthesis (3′-aminooxyalkylated oligomers) or formation of its phosphoramidite 6 followed by coupling with desired oligomer (for generating 5′-aminooxyalkylated oligomers). Both the routes produced modified oligonucleotides in sufficiently high yields and purity (on HPLC) via conventional oligonucleotide synthesis on an automated synthesizer and deprotection step using aqueous ammonia (16 h, 60 °C). Aminooxyalkylated oligonucleotides were used to construct microarrays on glass surface (biochips). The performance of the biochips was evaluated by immobilizing modified oligonucleotides on epoxylated glass microslides under different sets of conditions with respect to pH, temperature and time. Further, the constructed microarrays were successfully used for detection of nucleotide mismatches and bacterial typhoid.     

A new approach to the synthesis of the 5'-deoxy-5'-methylphosphonate linked thymidine oligonucleotide analogues.

A new synthetic method for the preparation of the 5'-deoxy-5'-methylphosphonate linked thymidine oligonucleotides (5'-methylenephosphonate analogues) was developed. The method is based on the use of a phosphonate protecting group, 4-methoxy-1-oxido-2-picolyl, enabling intramolecular nucleophilic catalysis which together with the condensing agent, 2,4,6-triisopropylbenzenesulfonyl chloride, secures fast and efficient formation of the 5'-methylenephosphonate internucleosidic bonds. The produced protected oligomers were treated with thiophenol and triethylamine to remove the phosphonate protecting groups, cleaved from the solid support using concentrated aqueous ammonia, and purified by HPLC. Several thymidine oligonucleotide analogues with the chain length of up to 20 nucleotidic units, in which all internal 5'-oxygen atoms have been replaced by methylene groups directly bound to phosphorus, were synthesised using this methodology.     

Synthesis of dihydrothymidine and thymidine glycol 5'-triphosphates and their ability to serve as substrates for Escherichia coli DNA polymerase I

5,6-Dihydrothymidine 5'-triphosphate (DHdTTP) was synthesized by catalytic hydrogenation of thymidine 5'-triphosphate (dTTP). Thymidine glycol 5'-triphosphate (dTTP-GLY) was prepared by bromination of dTTP followed by treatment with Ag2O. The modified nucleotides were extensively purified by anion-exchange high-performance liquid chromatography (HPLC). Alkaline phosphatase digestion of DHdTTP and dTTP-GLY gave the expected products (5,6-dihydrothymidine and cis-thymidine glycol), the identities of which were confirmed by reverse-phase HPLC using authentic markers. HPLC analysis of the alkaline phosphatase digested DHdTTP revealed that DHdTTP was a mixture of C5 diastereoisomers [(5S)- and (5R)-DHdTTP]. Despite the significant distortion of the pyrimidine ring in DHdTTP, it was incorporated in place of dTTP during primer elongation catalyzed by Escherichia coli DNA polymerase I Klenow fragment. The rate of incorporation of DHdTTP was about 10-25-fold lower than that of dTTP. On the other hand, dTTP-GLY, which also has a distorted pyrimidine ring, did not replace dTTP, and no elongation of the primer was observed. In order to study the preference of incorporation of the diastereoisomers of DHdTTP into DNA, salmon testes DNA, activated by exonuclease III, was used as a template for DNA polymerase I Klenow fragment in the presence of [3H]DHdTTP (S and R mixture) and normal nucleotides. After enzymatic digestion of the DNA to nucleosides, the products were analyzed by HPLC. The ratio of the isomers incorporated into DNA (S:R = 73.27) was virtually the same as that of the [3H]DHdTTP substrates (S:R = 79.21).(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis and purification of synthetic oligonucleotides by reversed-phase high-performance liquid chromatography with photodiode array and mass spectrometry detection.

Native and modified synthetic oligonucleotides were purified by reversed-phase HPLC using volatile ion-pairing mobile phases. Purification of 10-90 nmol of oligonucleotides in a single injection was demonstrated using a 4.6 x 75-mm HPLC column packed with porous 2.5 microm C18 sorbent. Separation of target products from N-1 failure fragments was achieved for oligonucleotides in the 4- to 60-mer size range. We employed a combination of absorbance and mass spectrometry detection to identify by-products of oligonucleotide synthesis. This method was also employed for analysis and purification of fluorescently labeled oligonucleotides.     

Photocleavable biotin phosphoramidite for 5'-end-labeling, affinity purification and phosphorylation of synthetic oligonucleotides.

We report the design, synthesis and evaluation of a non-nucleosidic photocleavable biotin phosphoramidite (PCB-phosphoramidite) which provides a simple method for purification and phosphorylation of oligonucleotides. This reagent introduces a photocleavable biotin label (PCB) on the 5'-terminal phosphate of synthetic oligonucleotides and is fully compatible with automated solid support synthesis. HPLC analysis shows that the PCB moiety is introduced predominantly on full-length sequences and is retained during cleavage of the synthetic oligonucleotide from the solid support and during subsequent deprotection with ammonia. The full-length 5-PCB-labeled oligonucleotide can then be selectively isolated from the crude oligonucleotide mixture by incubation with immobilized streptavidin. Upon irradiation with 300-350 nm light the 5'-PCB moiety is cleaved with high efficiency in <4 min, resulting in rapid release of affinity-purified 5'-phosphorylated oligonucleotides into solution. 5'-PCB-labeled oligonucleotides should be useful in a variety of applications in molecular biology, including cassette mutagenesis and PCR. As an example, PCB-phosphoramidite has been used for the synthesis, purification and phosphorylation of 50-and 60mer oligonucleotides.     

Fluorescent oligonucleotides and deoxynucleotide triphosphates: preparation and their interaction with the large (Klenow) fragment of Escherichia coli DNA polymerase I

Fluorescent derivatives of short oligonucleotides of defined sequence were prepared by the incorporation of 5-(propylamino)uridine via current phosphoramidite chemistry, followed by derivatization of the propylamine function with mansyl chloride. These oligomers, annealed to complementary oligomers, yielded short duplex DNA fluorescently labeled at a specific base. The fluorescence emission from this labeled duplex increases upon binding to the Klenow fragment of DNA polymerase I (KF) at specific positions within the duplex DNA. By varying the position of the label within the duplex DNA and observing the emission, points of strong enzyme-DNA interactions were elucidated. A similar fluorescent derivative of a deoxynucleoside triphosphate (dNTP), 5-[[[[[[(5- sulfonaphthalenyl)amino]ethyl]amino]carbonyl]- methyl]thio]-2'-deoxyuridine 5'-triphosphate (AEDANS-S-dUTP), was synthesized, whose emission also was increased upon binding to KF. The change in emission intensities between unbound and bound substrates enabled the measurements of KDs for the DNA and dNTP derivative, which were found to be 0.15 nM and 2.9 microM, respectively. Stopped-flow measurements on these species yielded association and dissociation rates for each. Anisotropy measurements of the labeled base at various positions in the duplex yielded values that support the measurements made by observing the emission intensities.     

One-pot enzymatic synthesis of UDP-D-glucose from UMP and glucose-1-phosphate using an ATP regeneration system

Glucose-1-phosphate uridylyltransferase from E. coli K12 was used to convert uridine-5'-triphosphate and glucose-1-phosphate to UDP-D-glucose. The conversion was efficient and completed within 5 minutes under the employed conditions. In addition, thymidine-5'-monophosphate kinase and acetate kinase were proven to be non-specific, converting udridine-5'-monophosphate to uridine-5'-triphosphate with 55% conversion after 6 h, which was much slower than the production of TTP under the same conditions (complete conversion within one hour). Since these two reactions could proceed under the same conditions, a one-pot synthesis of UDP-D-glucose with ATP regeneration was designed from easily available starting materials, and conversion up to 40% by HPLC peak integration was achieved given a reaction time of 4 h.     

The quantitative determination of metabolites of 6-mercaptopurine in biological materials. VII. Chemical synthesis by phosphorylation of 6-thioguanosine 5'-monophosphate, 5'-diphosphate and 5'-triphosphate, and their purification and identification by reversed-phase/ion-pair high-performance liquid chromatography and by various enzymatic assays

A fast and reliable two-step method has been established for the chemical synthesis of 6-thioguanosine 5'-monophosphate, 6-thioguanosine 5'-diphosphate and 6-thioguanosine 5'-triphosphate starting from the ribonucleoside. In the first step, 6-thioguanosine dissolved in triethyl phosphate, at high yield reacts with phosphorus oxide trichloride to 6-thioguanosine 5'-monophosphate which is purified by anion-exchange chromatography on DEAE-Sephadex using a step gradient of hydrochloric acid. In the second step, 6-thioguanosine 5'-monophosphate dissolved in water, reacts with phosphoric acid in the presence of pyridine/dicyclohexyl carbodiimide and is converted to 6-thioguanosine 5'-diphosphate and 6-thioguanosine 5'-triphosphate which are separated from each other and from the 6-thioguanosine 5'-monophosphate by anion-exchange chromatography on DEAE-Sephadex using a gradient of ammonium bicarbonate. Material from each step of the preparation procedure is separated by reversed-phase HPLC chromatography and analyzed for its free ribonucleoside content, 5'-monophosphate, 5'-diphosphate, 5'-triphosphate and small amounts of unidentified phosphorylated compounds. The purity of the final preparations and the identity of each 6-thioguanosine 5'-phosphate are proven by highly specific enzymatic peak-shifting/HPLC analyses using alkaline phosphatase, 5'-nucleotidase, pyruvate kinase, nucleoside diphosphate kinase and combined hexokinase/glucose 6-phosphate dehydrogenase.     

Selectivity of RNA chain initiation in vitro. 1. Analysis of RNA initiations by two-dimensional thin-layer chromatography of 5'-triphosphate-labeled oligonucleotides.

A method for the rapid and quantitative analysis of 5'-terminal oligonucleotides of RNAs made in vitro is described. The method involves synthesis of RNA in the presence of [gamma-32P]ATP or GTP, isolation of the RNA, and digestion with T1 or pancreatic ribonucleases to release labeled 5'-triphosphate termanated oligonucleotides. The oligonucleotides are then subjected to chromatography on a polyethyleniminecellulose thin-layer system using 2 M LiCl, 0.01 M EDTA (pH 6.5) in the first dimension and 1.5 M LiCl, 1.8 M formic acid, 0.005 M EDTA (pH 2.0) in the second. RNAs made with E. coli RNA polymerase and lambdacb2, T7, T4, and adenovirus 2 DNA yield characteristic fingerprint patterns. The utility of this method in studying selectivity of in vitro RNA chain initiation is discussed.     

Preparation of sensitive and specific oligonucleotide probes tailed using terminal transferase and dITP

An oligonucleotide probe tailed with deoxyadenosine-5'-triphosphate or deoxythymine-5'-triphosphate is detectable with high sensitivity, but has a major drawback--the tail co-hybridizes specifically to complementary sequences. This can be a problem when screening cDNA clones that contain poly(dA) sequences. While it is possible to mask the cDNA tail with unlabeled poly(dA) or poly(A) oligonucleotides, false-positive clones are still produced because complete masking of extremely long (dA) tails is difficult. As a result, only cDNA clones that have extremely long poly(dA) sequences are often obtained by hybridization screening using tailed probes. In this report, we describe an oligonucleotide probe tailed with DIG-labeled nucleotide in combination with deoxyinosine-5'-triphosphate that was highly specific and sensitive to cDNAs. Terminal deoxynucleotidyl transferase efficiently adds dI nucleotides to the 3'-end. The dI of the tails did not pair with any nucleotides under stringent hybridization so that the specificity of hybridization assays remained high without affecting the sensitivity of the test. Colony hybridization experiments demonstrated that there were very few (1 of 80 tested) false positives using this technique. Its use may increase the accuracy of cDNA screening.     

Substrate specificity of soluble mitochondrial ATPase]

The parameters of the hydrolysis of ATP and several analogs by soluble mitochondrial ATPase were determined. Vmax of the reaction decreases within the range: 2'-desoxy-ATP greater than ATP greater than etheno-ATP greater than GTP greater than 3'-O-methylATP greater than UTP. ATP, 2'-desoxypATP, 3'O-methyl-ATP, GTP, and etheno-ATP are hydrolysed by soluble mitochondrial ATPase with close Km(app) values. CTP is not hydrolysed by the enzyme and does not inhibit the ATPase reaction at a concentration of 10(-2) M. Nucleoside triphosphate derivatives with an "open" ribose cycle 9-[1',5'-dihydroxy-4-(S)-hydroxymethyl-3'-oxapent-2' (R)-yl]adenyl-5'-triphosphate, and 1-[1',5'-dihydroxy-4'-(S)-hydroxymethyl-3'-oxapent-2'(R)-yl[cytosine-5'-triphosphate are effective inhibitors of ATPase (Ki approximately 5.10(-5)M). Mitochondrial ATPase binds the ATP analogs that have hydrocarbon radicals-(CH2)2-, -(CH2)3-, and (CH2)4- instead of the ribose residues: 9-(2'hydroxyethyl)adenyl-2'-triphosphate, 9-(3'-hydroxypropyl)-adenine-3'-triphosphate, and 9-(4'-hydroxybutyl)adenine-4'-triphosphyl)adenine-4'-triphosphate were not hydrolysed by the enzyme, although they inbibit the ATPase reaction (Ki 2.10(-4)M). 9-(2'-hydroxyethyl)adenine-2'-triphosphate is hydrolysed by ATPase eight times more slowly than ATP. It is suggested that the hydrolysis of the substrates of mitochondrial ATPase is- preceded by the binding of the substrates in a tense conformation in the active site of the enzyme.     

Synthesis and properties of fluorescent NF-kappa B-recognizing hairpin oligodeoxyribonucleotide decoys

Intramolecular fluorescence quenching of cyanine dyes was investigated using a model hairpin oligonucleotide decoy encoding a NF-kappaB p50 subunit binding site. Two types of hairpin oligonucleotides were synthesized: (1) 5'-(6-aminohexyl)- and 3'-(3-aminopropyl)-linked (I); (2) 5'-(6-aminohexyl)- and 3'-[3-(3-hydroxypropyldithio)propyl]-linked (II). Oligonucleotide I was covalently modified using monofunctional either Cy3- or Cy5.5-N-hydroxysuccinimide esters. Using reverse-phase HPLC, mono-and dicyanineamide derivatives of I were isolated. Mono-Cy3-modified derivatives of I, but not the mono-Cy5.5-modified derivatives, showed a 2-fold higher Cy3 fluorescence intensity compared to the free dye. There was no detectable difference in fluorescence between the di-Cy3 derivative of I and the free dye at the same concentration. However, there was a 4-fold quenching of fluorescence in the case of the di-Cy5.5 derivative of the same hairpin oligonucleotide. The quenching of Cy5.5 fluorescence could not be explained by the interaction of Cy5.5 with nucleotide bases as demonstrated by incubating free Cy5.5 dye with oligonuclotides. The quenching effect was further investigated using an oligonucleotide bearing a cleavable 3'-amino-terminated linker bearing an S-S bond (III). After modification of the 5'- and 3'-end of oligonucleotide III with a Cy5.5 monofunctional hydroxysuccinimide ester, a 70-75% quenching of fluorescence was observed. Fluorescence was 100% dequenched after the reduction of S-S bond. The obtained result unequivocally demonstrates that the formation of intramolecular Cy5.5 dimers is the dominant mechanism of fluorescence quenching in symmetric dye-dye hairpin decoy beacons.     

Inhibition of primase, the dnaG protein of Escherichia coli by 2'-deoxy-2'-azidocytidine triphosphate.

2'-Deoxy-2'-azidocytidine-5'-triphosphate was investigated as an inhibitor in two reconstructed enzyme systems which catalyze the replication of two viral DNAs. During replication of the duplex replicative form of phiX174 DNA, DNA polymerase III holoenzyme was weakly inhibited and inhibition was reversed by dCTP. A more pronounced inhibition, not reversed by either dCTP or CTP, was observed during replication of the single-stranded DNA of the bacteriophage G4, a close relative of phiX174. This effect depended on the incorporation of 2'-deoxy-2'-azidocytidine-5'-triphosphate by primase (dnaG protein) which synthesizes a 29-residue RNA primer at the unique origin of bacteriophage G4 DNA replication. Extension of the primer strand, terminated by 2'-deoxy-2'-azidocytidine-5'-triphosphate is then severely inhibited. Primase was also inhibited by the 2'-deoxy-2'-azido derivatives of ATP, GTP, and UTP.     

Preparation of DNA-duplexes, containing internucleotide thiophosphate groups in various positions of the recognition site for the EcoRII restriction endonuclease

Twenty-four 12-mer DNA duplexes, each containing a chiral phosphorothioate group successively replacing one of the internucleotide phosphate groups either in the EcoRII recognition site (5'CCA/TGG) or near to it, were obtained for studying the interaction of the restriction endonuclease EcoRII with internucleotide DNA phosphates. Twelve of the 12-mer oligonucleotides were synthesized as Rp and Sp diastereomeric mixtures. Six of them were separated by reversed-phase HPLC using various buffers. Homogeneous diastereomers of the other oligonucleotides were obtained by enzymatic ligation of the Rp and Sp diastereomers of 5- to 7-mer oligonucleotides preliminarily separated by HPLC with the corresponding short oligonucleotides on a complementary DNA template. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2003, vol. 29, no. 6; see also http://www.maik.ru.     

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.     

The preparation and application of functionalised synthetic oligonucleotides: III. Use of H-phosphonate derivatives of protected amino-hexanol and mercapto-propanol or -hexanol.

Syntheses of H-phosphonate salts (4a-e) of N/S-protected alcohols such as 6-aminohexan-1-ol, 3-mercaptopropan-1-ol and 6-mercaptohexan-1-ol are described using 2-chloro-5,6-benzo-1,3,2-phosphorin-4-one (2) as the phosphonylating agent. The H-phosphonate salts (4a-e), in the presence of pivaloyl chloride or adamantoyl chloride as an activator, were coupled to the 5'-end of synthetic oligonucleotides on solid supports to produce amino or thio-linked oligonucleotides. Following deprotection and purification, fluorescent dyes, biotin derivatives and poly-L-lysine-maleimide were separately attached to the functionalised oligonucleotides. Identical derivatized oligomers were obtained with cyanoethyl-N,N-diisopropylamidite chemistry and amidites (5a-e) of the respective alcohols.     

Analysis of DNA-microarrays produced by inverse in situ oligonucleotide synthesis.

5'-Phosphoramidites protected by 2-nitrophenylethyl (NPE) and 2-(4-nitrophenyl)ethoxy carbonyl (NPEOC) functions were employed for in situ synthesis of oligonucleotides in 5'-->3' direction on flat glass surfaces. By this inverse synthesis format, the oligonucleotides are attached to the solid support via their 5'-ends while the free 3'-hydroxyl groups are available as substrates for enzymatic reactions such as elongation by polymerases, thereby adding another feature to the portfolio of chip-based applications. Having a fluorescence dye present at the first base during synthesis, the quality of the oligonucleotides was analysed quantitatively by capillary electrophoresis after release from the solid support. With about 95% yield per condensation, it was found to be equivalent to synthesis results achieved on CPG support. The chip-bound oligonucleotides could be extended enzymatically upon hybridisation of a DNA-template. Surprisingly, however, only 63% of the oligonucleotides were elongated in polymerase reactions, while oligonucleotides that were released from the support behaved normally in standard PCR amplifications. This rate of 63% nevertheless compares favourably with an extension rate of only 50%, which was achieved under identical conditions, if pre-fabricated oligonucleotides of identical sequence had been spotted to the glass support.     

Analysis of reducing sugars as their chromophoric hydrazones by high-performance liquid chromatography

A chromophoric hydrazide, 4'-N,N-dimethylamino-4-azobenzene sulfonyl hydrazide (DABS-hydrazide), was prepared from 4'-N,N-dimethylamino-4-azobenzene sulfonyl chloride by reaction with hydrazine. Reducing sugars were derivatized with DABS-hydrazide at 50 degrees C for 120 min. The chromophoric hydrazones were separated by reversed-phase HPLC isocratically using a short column (4.6 X 50 mm) and 0.08 M acetic acid-acetone as an eluant with no sample pretreatment and were quantitated at the picomole level. This method was applied to the sugar analysis of 5 micrograms of glycoproteins. Dansyl hydrazide derivatives of sugars were also separated by this HPLC system.     

Cyanamide mediated syntheses under plausible primitive earth conditions

When an aqueous solution (pH 7.0) of 3H deoxythymidine 5'-triphosphate, deoxythymidine 5'-phosphate, 4-amino-5-imidazolecarboxamide, cyanamide and ammonium chloride was dried and heated at 60 degrees C for 18 h, oligomers were obtained in a yield of approximately 80%. After the chemical degradation of any pyrophosphate bonds present in these oligomers, linear polynucleotides of up to 7-8 units in length were isolated by DEAE cellulose column chromatography and identified by enzymatic digestion procedures. The di- and trinucleotide fractions were degraded 87% and 100% by snake venom phosphodiesterase and 39% and 9% by spleen phosphodiesterase. This synthesis of deoxythymidine oligonucleotides was conducted under potentially prebiotic conditions and may offer a possible method for the synthesis of deoxyoligonucleotides on the primitive Earth.