Quantitative Bestimmung von Desoxyribonucleosid-Triphosphaten in Ascitestumorzellen unter Verwendung von 32P

Zusammenfassung Von Ascitestumoren wurden nach Inkubation mit ³²P-Orthophosphat Extrakte der säurelöslichen Verbindungen hergestellt und dünnschichtchromatographisch getrennt. Aus dem ³²P-Gehalt auf den durch die Verwendung von Trägersubstanzen lokalisierbaren Positionen der Desoxyribonucleosid-Triphosphate und der spezifischen Aktivität des innercellulären Orthophosphats wurden die in den Zellen vorliegenden Gehalte an Desoxyribonucleosid-Triphosphaten ermittelt.

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Quantitative determination of deoxyribonucleoside triphosphates in ascites tumor cells by using ³²P

The acid soluble compounds of ascites tumor cells after incubation with ³²P-orthophosphate were separated by thin-layer chromatography. By using unlabelled deoxyribonucleoside triphosphates it was possible to localize the corresponding radioactive spots. From the radioactivity of the spots and the specific activity of the intracellular orthophosphate the amounts of the deoxyribonucleoside triphosphates could be calculated.

     

An Optimized Ion-Pair HPLC Method for Simultaneous Analysis of Nucleoside Triphosphate Levels in Hepatoma Cell Line

An improved ion-pair HPLC method was developed for the simultaneous determination of ribonucleoside triphosphates and their corresponding deoxyribonucleoside triphosphates in HepG2 cell extracts. HPLC conditions, flow rate and column temperature were optimized and good linearity (r ²  > 0.9993) was obtained over the investigated concentration ranges. Reproducibility was evaluated by intra- and inter-day assays and RSD values were below 5.39%. Recoveries ranged from 98.2 ± 3.49% to 103.1 ± 1.75%, respectively. Finally, the method was successfully applied to the analysis of eight compounds present in HepG2 cell extracts.     

Improved sample preparation method for high-performance liquid chromatography of deoxyribonucleoside triphosphates from cell culture extracts

The accurate determination of deoxyribonucleoside triphosphates in cells is difficult owing to the high concentrations of interfering ribonucleoside triphosphates. The latter can be degraded to their respective bases by periodate oxidation of cell extracts. However, the large amount of bases so produced can interfere with subsequent high-performance liquid chromatographic (HPLC) analysis. The use of a weak ion-exchange cartridge to partially purify and concentrate deoxyribonucleoside triphosphates in periodate-treated cell extracts, prior to HPLC, thus allowing accurate determination is described. The recovery of the deoxyribonucleoside triphosphates is greater than 95%, and greater than 90% of the interfering bases are removed.     

A simplified method for the preparation of α-32P-labeled deoxyribonucleoside triphosphates

A method for preparing α-³²P-labeled deoxyribonucleoside triphosphates ([α-³²P]dNTP)^* is described. By comparing the labeling procedure to that ofWalseth et al.^1,2 the reaction of Nuclease P₁ is saved in our experiments and only three steps are needed for a routine preparation, without decreasing the yield of [α-³²P]dNTP which is over 92% based on³²Pi. In this paper [α-³²P]dATP is taken as an example to deseribe the labeling procedure, the suitability of our method has been discussed.     

Antitumor Mechanism of Hydroxycamptothecin via the Metabolic Perturbation of Ribonucleotide and Deoxyribonucleotide in Human Colorectal Carcinoma Cells

Hydroxycamptothecin (SN38) is a natural plant extract isolated from Camptotheca acuminate. It has a broad spectrum of anticancer activity through inhibition of DNA topoisomerase I, which could affect DNA synthesis and lead to DNA damage. Thus, the action of SN38 against cancers could inevitably affect endogenous levels of ribonucleotide (RNs) and deoxyribonucleotide (dRNs) that play critical roles in many biological processes, especially in DNA synthesis and repair. However, the exact impact of SN38 on RNs and dRNs is yet to be fully elucidated. In this study, we evaluated the anticancer effect and associated mechanism of SN38 in human colorectal carcinoma HCT 116 cells. As a result, SN38 could decrease the cell viability and induce DNA damage in a concentration-dependent manner. Furthermore, cell cycle arrest and intracellular nucleotide metabolism were perturbed due to DNA damage response, of which ATP, UTP, dATP, and TTP may be the critical metabolites during the whole process. Combined with the expression of deoxyribonucleoside triphosphates synthesis enzymes, our results demonstrated that the alteration and imbalance of deoxyribonucleoside triphosphates caused by SN38 was mainly due to the de novo nucleotide synthesis at 24 h, and subsequently the salvage pathways at 48 h. The unique features of SN38 suggested that it might be recommended as an effective supplementary drug with an anticancer effect.     

Potentiometric Characterization of Telomerase Activity Using a Copper(ii)-pyrophosphate Complex with a Copper ion-selective Electrode

A novel potentiometric sensing platform was designed for real-time electronic monitoring of telomerase activity using a copper(II) ion-selective electrode (Cu-ISE). The target-induced coordination of pyrophosphate ion with copper ion (Cu²⁺) was utilized for the detection of the enzyme activity. Upon telomerase introduction, a DNA primer was elongated in the presence of deoxyribonucleoside triphosphates, accompanying formation of the by-product (pyrophosphate ion; P₂O₇^4-). The generated pyrophosphate ions chemically coordinated with free copper ions to form the copper(II)-pyrophosphate complex, thereby resulting in a decrease in the number of free copper ions present in solution. With the addition of telomerase, the electrode potential on the Cu-ISE increased relative to the background signal. The results indicated that the copper(II)-pyrophosphate system exhibited good potentiometric response for the detection of telomerase activity, and allowed the determination of the analyte in HeLa cell extract at concentrations as low as 36 cells mL^?1. Moreover, the Cu-ISE-based sensing platform afforded good reproducibility, thus representing a useful approach for practical use in quantitative telomerase activity assays.     

Synthesis of Aldehyde‐Linked Nucleotides and DNA and Their Bioconjugations with Lysine and Peptides through Reductive Amination

5‐(5‐Formylthienyl)‐, 5‐(4‐formylphenyl)‐ and 5‐(2‐fluoro‐5‐formylphenyl)cytosine 2′‐deoxyribonucleoside mono‐ ( dC^RMP ) and triphosphates ( dC^RTP ) were prepared by aqueous Suzuki–Miyaura cross‐coupling of 5‐iodocytosine nucleotides with the corresponding formylarylboronic acids. The dC^RTP s were excellent substrates for DNA polymerases and were incorporated into DNA by primer extension or PCR. Reductive aminations of the model dC^RMP s with lysine or lysine‐containing tripeptide were studied and optimized. In aqueous phosphate buffer (pH 6.7) the yields of the reductive aminations with tripeptide III were up to 25 %. Bioconjugation of an aldehyde‐containing DNA with a lysine‐containing tripeptide was achieved through reductive amination in yields of up to 90 % in aqueous phosphate buffer.     

Protection-free one-pot synthesis of 2'-deoxynucleoside 5'-triphosphates and DNA polymerization

By differentiating the functional groups on nucleosides, we have designed and developed a one-pot synthesis of deoxyribonucleoside 5'-triphosphates without any protection on the nucleosides. A facile synthesis is achieved by generating an in situ phosphitylating reagent that reacts selectively with the 5'-hydroxyl groups of the unprotected nucleosides. The synthesized triphosphates are of high quality and can be effectively incorporated into DNAs by DNA polymerase. This novel approach is straightforward and cost-effective for triphosphate synthesis.     

7‐Aryl‐7‐deazaadenine 2′‐Deoxyribonucleoside Triphosphates (dNTPs): Better Substrates for DNA Polymerases than dATP in Competitive Incorporations

A series of 7‐substituted 7‐deazaadenine and 5‐substituted cytosine 2′‐deoxyribonucleoside triphosphates (dNTPs) were tested for their competitive incorporations (in the presence of dATP and dCTP) into DNA by several DNA polymerases by using analysis based on cleavage by restriction endonucleases. 7‐Aryl‐7‐deazaadenine dNTPs were more efficient substrates than dATP because of their higher affinity for the active site of the enzyme, as proved by kinetic measurements and calculations.     

Synthesis of 2′-Deoxyisoguanosine 5′-Triphosphate and 2′-Deoxy-5-methylisocytidine 5′-Triphosphate

The syntheses of the 5′-triphosphates of 2′-deoxyisoguanosine (=p₃isoG_d) and 2′-deoxy-5-methylisocytidine (=p₃me⁵isoC_d), two new bases for the genetic alphabet, are described. The triphosphates were synthesized from the corresponding nucleosides using a transient-protection procedure. The introduction of a methyl group at the 5-position of 2′-deoxyisocytidine remarkably improved the stability of the triphosphate. Characterization of the triphosphates included enzymatic incorporation opposite the complementary base in a template oligonucleotide.     

Incorporation of a minimal nucleotide into DNA

Abasic sites are amongst the most frequent DNA lesions and result from spontaneous hydrolysis of the glycosidic bond or from the removal of damaged nucleobases. These depurination events can also occur on free deoxyribonucleoside triphosphates present in cells and lead to the formation of an abasic site triphosphate of which very little is known. Herein, we report the synthesis and biochemical characterization of the minimal triphosphate dФTP. Unexpectedly, dФTP is tolerated by various DNA polymerases and the incorporation efficiency obeys the A-rule. Single incorporation of dФMP units were also observed opposite abasic sites and the addition of prosthetic molecules mimicking base-pairs do not seem to favor the process.     

Zur Bestimmung von β-Aminocrotonsäureester nach Migration in Lebensmittel

β-Aminocrotonic esters are of great importance as stabilizers for the production of clearly transparent food packaging of PVC. For the purpose of studying the migration into foods a thin-layer Chromatographic and a polarographic method were elaborated. The TLC method consists of the visual comparison of the intensity of the spots after treatment with Fast Blue B salt. The polarographic determination is carried out after nitrosation of the stabilizer. By the TLC method 10^?7 g of ester per spot are still detectable; the concentration which is still determinable by cathode-ray polarography is 5×10^?7 g of ester and by conventional d.c. polarography 5×10^?6g of ester per ml of final solution. After extraction with acetonitrile traces of the stabilizer which are migrated into edible oil are still determinable down to 2 ppm by the methods described.     

Total nucleotide analysis of Hydra DNA and RNA by MEKC with LIF detection and 32P‐postlabeling

The model organism Hydra has been used for molecular studies for more than 20 years, however, its DNA base composition has not been determined yet. We have analyzed DNA and total RNA of the freshwater polyp Hydra magnipapillata with two independent procedures of high accuracy and sensitivity – fluorescence labeling of nucleotides followed by CE‐LIF detection and ³²P‐postlabeling. DNA of Hydra was digested either to deoxyribonucleoside‐5′‐monophosphates or deoxyribonucleoside‐3′‐monophosphates selectively conjugated with the fluorescent dye 4,4‐difluoro‐5,7‐dimethyl‐4‐bora‐3a,4a‐diaza‐s‐indacene‐3‐propionyl ethylene diamine hydrochloride (BODIPY FL EDA) separated and detected using CE‐LIF. Both versions of the assay revealed a high A+T composition of 78 and 71%, whereas total DNA methylation (5‐methyldeoxycytidine) was 2.6 and 3.1%. Total Hydra RNA showed highest base levels for guanine (33%) and a level of 1.4% for pseudouracil. All values were in good agreement with those determined by the ³²P‐postlabeling method.     

Radiodünnschichtchromatographische Spurenbestimmung von Dithionsäure

A modified method of isotope dilution analysis is described for the determination of small amounts of dithionic acid (1–10 μg). The conditions for the setting up of the calibration curve are reported. The inactive dithionic acid is labelled with³⁵S compound. After separation by thin layer chromatography on extremely thin layers the activity profiles of the spots are measured. The reduced maximal intensities are plotted against the amount of dithionic acid. The calibration curve is also valid for solutions containing up to 1M of sulphuric acid.     

Synthesis of Isonucleoside 5′-Triphosphates

Abstract

A series of isonucleoside 5′-triphosphates were synthesized via a rapid and efficient method. The structures of the triphosphates (8a–8d) were characterized by ³¹P NMR and TOF-MS.     

Aerolysin蛋白纳米孔道直接检测单个DNA碱基修饰

基于Aerolysin生物膜通道蛋白的纳米孔道电化学分析技术,因其高的电化学空间限域能力可实现超灵敏DNA单分子检测。本文利用单个Aerolysin纳米孔道在无需标记、无需扩增的条件下直接分辨3种具有单个碱基差异的单链DNA。实验结果显示,具有单个炔基侧链基团修饰的单个ss DNA在限域空间内与Aerol-ysin纳米孔道的相互作用时间较未修饰的ss DNA增长近7倍,电流阻断程度增大7%,且高斯峰半峰宽减小了44%,增强了Aerolysin纳米孔道对单个DNA分子的分辨能力。研究成果有望推动Aerolysin纳米孔在DNA直接测序及表观遗传修饰检测中的应用。     

Polymer Supported Synthesis of Deoxyoligonucleotides using In Situ Prepared Deoxynucleoside 2-Cyanoethyl Phosphoramidites

Abstract

2-Cyanoethyl bis(diisopropylamino) phosphorodiamidite (1) is a stable and easily obtainable compound¹ which, when activated with 0.5 eq. tetrazole, gives solutions of deoxyribonucleoside 2-cyano-ethyl phosphoramidites (2) useful for the synthesis of deoxyoligo-nucleotides. ² Our results applying these in-situ prepared phosphoramidites for polymer-supported syntheses of a variety of deoxyoligonucleotides (DNA fragments) will be presented.     

Die chromatographische Analyse von Phosphaten

Zusammenfassung Durch vergleichende quantitative Untersuchungen wird festgestellt, daß die Verwendung von Methanol-Fließmitteln zur papierchromatographischen Trennung von kondensierten Phosphaten zu einer starken Hydrolyse insbesondere von Triphosphaten führt. Als Hydrolyseprodukt entsteht hauptsächlich Diphosphat und nicht, wie zu erwarten, Mono-und Diphosphat im stöchiometrischen Verhältnis.Es wird ein Dioxan-Fließmittel mitgeteilt, das eine optimale und weitestgehend hydrolysefreie Auftrennung kondensierter Phosphate in Einzelzonen von n = 1–10 und n > 10 gestattet und eine Arbeitsanweisung zur quantitativen papierchromatographischen Bestimmung kondensierter Phosphate gegeben.Darüber hinaus wird gezeigt, daß bei der Papierchromatographie kondensierter Phosphate neben starken Flecken der Hauptbestandteile bereits Spuren anderer Kondensationsgrade als Einzelflecken identifiziert werden können, deren P₂O₅-Anteil jedoch außerhalb der Bestimmbarkeit in der papierchromatographischen quantitativen Analyse liegt.Ferner wird der Nachweis geführt, daß in handelsüblichen Natriumtriphosphaten auch höher linear kondensierte Polyphosphate enthalten sein können.

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Summary By comparing quantitative examinations it is established that the use of methanol solvent mixtures for the separation of condensed phosphates by paper chromatography causes a considerable hydrolysis especially of triphosphates. As product of hydrolysis mainly diphosphate is formed and not—as expected—mono-and diphosphate in stoichiometric proportion.A dioxan solvent mixture is described permitting an optimum separation of condensed phosphates in single bands from n = 1 to 10 and n > 10 nearly free of hydrolysis and a detailed instruction is given for the quantitative paper chromatography of condensed phosphates.Further, it is shown that on a paper chromatogram of condensed phosphates besides the strong spots of the main constituents also traces of other degrees of condensation may be identified, which, however, are below the limit of quantitative detection of paper-chromatographic methods.Moreover, it is pointed out that in commercial sodium triphosphate also higher linearly condensed phosphates may be contained.

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I. Teil: Rössel, T.: diese Z. 196, 6 (1963). II. Teil: Rössel, T.: diese Z. 197, 333 (1963).     

Alkylsulfanylphenyl derivatives of cytosine and 7-deazaadenine nucleosides, nucleotides and nucleoside triphosphates: synthesis, polymerase incorporation to DNA and electrochemical study

Aqueous Suzuki–Miyaura cross‐coupling reactions of halogenated nucleosides, nucleotides and nucleoside triphosphates derived from 5‐iodocytosine and 7‐iodo‐7‐deazaadenine with methyl‐, benzyl‐ and tritylsufanylphenylboronic acids gave the corresponding alkylsulfanylphenyl derivatives of nucleosides and nucleotides. The modified nucleoside triphosphates were incorporated into DNA by primer extension by using Vent(exo‐) polymerase. The electrochemical behaviour of the alkylsulfanylphenyl nucleosides indicated formation of compact layers on the electrode. Modified nucleotides and DNA with incorporated benzyl‐ or tritylsulfanylphenyl moieties produced signals in [Co(NH₃)₆]³⁺ ammonium buffer, attributed to the Brdi?ka catalytic response, depending on the negative potential applied. Repeated constant current chronopotentiometric scans in this medium showed increased Brdi?ka catalytic response, which suggests the deprotection of the alkylsulfanyl derivatives to free thiols under the conditions.