A simple and highly sensitive DNAzyme-based assay for nicotinamide adenine dinucleotide by ligase-mediated inhibition of strand displacement amplification

Existing strategies for detecting nicotinamide adenine dinucleotide (NAD⁺) or other cofactors are commonly cumbersome and moderate sensitive. We report a novel DNAzyme-based visual assay strategy for NAD⁺ based on ligase-mediated inhibition of the strand displacement amplification (SDA). In the presence of NAD⁺, the SDA can be inhibited by the ligase reaction of two primers, which can initiate the SDA reaction in the case of no ligation, resulting in a dramatically decreasing yield of the SDA product, a G-quadruplex DNAzyme that can quantitatively catalyze the formation of a colored product. Therefore, the quantitative analysis for NAD⁺ can be achieved visually with high sensitivity. The developed strategy provides a simple colorimetric approach with high selectivity against most interferences and a detection limit as low as 50 pM. It also provides a universal platform for investigating cofactors or other related small molecules as well as quantifying the activity of DNA ligases.     

Sample preparation workflow for the liquid chromatography tandem mass spectrometry based analysis of nicotinamide adenine dinucleotide phosphate cofactors in yeast†

The accurate quantification of the highly unstable intracellular cofactor nicotinamide adenine dinucleotide phosphate in its oxidized and reduced forms demands a thorough evaluation of the analytical workflow and dedicated methods reflecting their solution chemistry as well as the biological importance of their ratio. In this work, we present a workflow for the analysis of intracellular levels of oxidized and reduced nicotinamide adenine dinucleotide phosphate in the yeast Pichia pastoris, including hot aqueous extraction, chromatographic separation in reversed‐phase conditions employing a 100% wettable stationary phase, and subsequent tandem mass spectrometric analysis. A thorough evaluation and optimization of the sample preparation procedure resulted in excellent biological repeatabilities (on average <10%, N = 3) without employing an internal standardization approach. As a consequence, the methodology proved to be appropriate for the relative assessment of intracellular levels of oxidized and reduced nicotinamide adenine dinucleotide phosphate in different P. pastoris strains. The ratio of reduced versus oxidized nicotinamide adenine dinucleotide phosphate was significantly higher in an engineered strain overexpressing glucose‐6‐phosphate dehydrogenase than in the corresponding wildtype strain. Interestingly, a difference was also observed in the nicotinamide adenine dinucleotide phosphate pool size, which was significantly higher in the wildtype than in the modified strain.     

Fabrication and characterization of Meldola's blue/zinc oxide hybrid electrodes for efficient detection of the reduced form of nicotinamide adenine dinucleotide at low potential

We report the synthesis and the electrochemical properties of hybrid films made of zinc oxide (ZnO) and Meldola's blue dye (MB) using cyclic voltammetry (CV). MB/ZnO hybrid films were electrochemically deposited onto glassy carbon, gold and indium tin oxide-coated glass (ITO) electrodes at room temperature (25 ± 2 °C) from the bath solution containing 0.1 M Zn(NO₃)₂, 0.1 M KNO₃ and 1 × 10⁻⁴ M MB. The surface morphology and deposition kinetics of MB/ZnO hybrid films were studied by means of scanning electron microscopy (SEM), atomic force microscopy (AFM) and electrochemical quartz crystal microbalance (EQCM) techniques, respectively. SEM and AFM images of MB/ZnO hybrid films have revealed that the surfaces are well crystallized, porous and micro structured. MB molecules were immobilized and strongly fixed in a transparent inorganic matrix. MB/ZnO hybrid films modified glassy carbon electrode (MB/ZnO/GC) showed one reversible redox couple centered at formal potential (E⁰′) −0.12 V (pH 6.9). The surface coverage (Γ) of the MB immobilized on ZnO/GC was about 9.86 × 10⁻¹² mol cm⁻² and the electron transfer rate constant (ks) was determined to be 38.9 s⁻¹. The MB/ZnO/GC electrode acted as a sensor and displayed an excellent specific electrocatalytic response to the oxidation of nicotinamide adenine dinucleotide (NADH). The linear response range between 50 and 300 μM NADH concentration at pH 6.9 was observed with a detection limit of 10 μM (S/N = 3). The electrode was stable during the time it was used for the full study (about 1 month) without a notable decrease in current. Indeed, dopamine (DA), ascorbic acid (AA), acetaminophen (AP) and uric acid (UA) did not show any interference during the detection of NADH at this modified electrode.     

生化教材中存在44年的瑕疵——吡啶环上氮原子的化合价

Understanding redox reactions in reaction of nicotinamide adenine dinucleotide (NAD⁺) and nicotinamide adenine dinucleotide phosphate (NADP⁺) requires a clear grasp of the textbook content. The reactions in nicotinamide coenzymes with reduced and oxidized forms have been compared in various biochemistry textbooks. Incorrect interpretations usually emphasize the valence changes that at nitrogen in the pyridine ring of a nicotinamide from +5 to +3. Actually, the valence of nitrogen in pyridine ring is -3. We have gathered shreds of evidences and provide here possible suggestions and caution for readers and instructors.     

Inhibition of the nicotinamide adenine dinucleotide-oxidoreductase reaction by herbicides and fungicides of various structures

The effect of herbicides (basagran, zenkor, kusagard, roundup, setoxidim, and lontrel and lontrel complexes with some doubly charged metal ions) and fungicides (tachigaren and tilt) on the activity of nicotinamide adenine dinucleotide (NADH)-oxidoreductase from the methylotroph Methylococcus capsulatus (strain M) was studied. All the herbicides and fungicides inhibit the enzyme, differing in the degree and type of inhibition. The inhibition constants K _i for these compounds and for lontrel complexes were determined. A correlation between the K _i values and the complexation constants of these pesticides with NADH was established. The studied compounds are toxic. __________ Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 5, pp. 1284–1289, May, 2005.     

Photolytic oxidation of reduced nicotinamide adenine dinucleotide

A photo-reaction between thionine dye and NADH has been observed which leads to the oxidation of the co-factor. Thionine (fluorescent), which is converted into semi/leuko thionine (non-fluorescent) during light reaction, fully recovers during the dark reaction. Analytical applications of this discovery are discussed. This reaction opens the door to the measurement of NADH using the fluorescence quenching of thionine and to the construction of several optical biosensors.     

痕量还原性辅酶Ⅰ的抑制荧光法测定

基于烟酰胺腺嘌呤二核苷酸 (简称辅酶I ,NADH)对血红蛋白催化H2 O2 氧化L 酪氨酸的反应具有强烈的抑制作用 ,提出了一种新的测定NADH的荧光分析法。该方法灵敏 ,简单 ,其线性范围为 5 .0× 1 0 -8～2 .0× 1 0 -6mol/L ;检出限为 2 .0× 1 0 -8mol/L。进一步探讨了NADH对血红蛋白催化反应的抑制机理 ,其抑制类型属于竞争性抑制     

Thermodynamics of redox reactions involving nicotinamide adenine dinucleotide

Literature data on the thermodynamics of redox nicotinamide adenine dinucleotide (NAD) dependent reactions have been analyzed. It has been established that for the redox reaction of NAD

where all substances except H₂ are in the aqueous buffer with the ionization enthalpy equal to zero, the most reliable thermodynamic parameters should be considered as: ΔH(298.15 K; pH 7)=?27.4±1.7 kJ mole^?1; ΔG (298.15K; pH 7)=±17.8 kJ mole^?1. From the above thermodynamic parameters of the reaction ΔH, ΔG and ΔS for reactions of NAD with natural substrates, synthetic mediators and some inorganic compounds have been calculated.     

Studies on Synthesis and Chromatographic Properties of NAD Covalently Bound onto Phospholipid-Coated Aminated Silica

Abstract

Recently affinity chromatography with a biospecific stationary phase was widely used in separation and purification processes of all kinds of enzymes. In this paper, a series of synthetic reactions of solid-liquid phase on a silica surface are described. By using a wide-pore (30μm). microspheric silica (8μm) as the matrix and γ-aminopropyltriethoxy-silane as the activating agent, the nicotinamide adenine dinucleotide (NAD) was bonded through its amino groups with carboxylic groups of linked phospholipid by a covalent bond on the aminated supports. This bonded stationary phase provided high thermal stability which could be used for separating nucleotides with good resolution. At the same time, the effects of pH, organic modifier and ionic strength on the retention properties of nucleotides were also investigated.     

Fluorescence quenching of thionine by reduced nicotinamide adenine dinucleotide

Reduced nicotinamide adenine dinucleotide (NADH) is shown to quench the fluorescence of thionine. Quenching of thionine is extremely efficient with a half quenching concentration of only 16.1 × 10⁻⁶ M NADH. A Stern—Volmer plot is linear over the NADH concentration range from 1 to 20μM. The corresponding Stern—Volmer quenching constant is 6.2 × 10⁴ M⁻¹ and the limit of detection for NADH measurements is 1.6 × 10⁻⁶ M. Process of quenching is attributed to the formation of an exciplex between thionine and NADH. Potential analytical features of this system are discussed.     

Site-specific immobilization of flavin adenine dinucleotide on indium/tin oxide electrodes through flavin adenine amino group

A Mannich-type reaction was used to attach flavin adenine dinucleotide (FAD) covalently to aminosilane derivatized indium/tin oxide-coated glass plates. The aminosilane was activated with formaldehyde to give an intermediate that attached specifically to the adenine amino group of FAD. The presence of the intermediate also was demonstrated by coupling hydroquinone to the formaldehyde activated support. The immobilized FAD and hydroquinone were characterized by cyclic or differential pulse voltammetry. The immobilized FAD was shown to reduce the overpotential for NADH oxidation by 180 mV. In keeping with results for FAD on glassy carbon, FAD attached to indium/tin oxide at the adenine amino group did not lead to reconstitution of activity with apoglucose oxidase. On leave from University of Madras, India.     

烟酰胺腺嘌呤二核苷酸磷酸与生物光化学

生物光化学的核心内容是光合作用。在光合作用这个生物光化学的核心领域，烟酰胺腺嘌呤二核苷酸磷酸(NADPH)始终起着极其重要的作用，NADPH就是一个传递电子和能量的最关键活性生物分子。本文对海洋里的光合作用、细菌与光合作用以及光合作用的模拟等作了介绍。     

Nicotinamide Adenine Dinucleotide Oxidation Studies at Multiwalled Carbon Nanotube/Polymer Composite Modified Glassy Carbon Electrodes

NADH oxidation has previously been investigated at carbon nanotube surfaces, although studies into the effect of the polymer binders are needed to fully understand whether the polymer binder affects the electrochemistry. This work details NADH oxidation at glassy carbon electrodes modified with composites containing multiwalled carbon nanotubes and selected polymer binders. NADH is shown to be oxidized at a lower potential than at glassy carbon electrodes and the oxidation potential is a function of the polymer binder. Hydrophobically modified Nafion, Nafion, linear poly(ethylenimine) (LPEI), octyl‐modified LPEI, and poly(vinylpyridine) binders were studied. Experiments showed the peak current and electrochemically assessible electrode area are dependent on the polymer binder. Overall, this paper shows that polymer binders affect NADH oxidation potential at carbon nanotube modified electrodes.     

Synthesis of Nicotinamide Adenine Dinucleotide (NAD) Analogues with a Sugar Modified Nicotinamide Moiety

The synthesis of nicotinamide adenine dinucleotide (NAD) analogues in which the ribose unit of the nicotinamide moiety is replaced by a hexitol, altritol, and cyclohexenyl sugar mimic is described.     

双稳定态化学振荡体系测定尼克酰胺腺嘌呤二核苷酸磷酸

利用酸性介质中Ce 催化溴酸钾 丙二酸BZ化学振荡体系的原理 ,建立了一种新型的测定尼克酰胺腺嘌呤二核苷酸磷酸 (NADP)的方法。报道了NADP的浓度在 1.0× 10 -8～ 1.0× 10 -6mol/L范围内的振荡体系中振幅与NADP浓度之间呈良好的线性关系 ,相关系数r为 0 .9999。对NADP参与的反应机理进行了探讨。     

Novel nicotinamide adenine dinucleotide analogues as selective inhibitors of NAD-dependent enzymes

Three novel dinucleotide analogues of nicotinamide adenine dinucleotide (NAD⁺) have been synthesised from d-ribonolactone. These compounds incorporate a thiophene moiety in place of nicotinamide and are hydrolytically stable. They have been evaluated as inhibitors of adenosine diphosphate ribosyl cyclase, glutamate dehydrogenase and Sir2 acyltransferase activities. Enzyme specificity and a high level of inhibition was observed for the dehydrogenase.     

On the nuclear magnetic resonance spectrum of reduced nicotinamide adenine dinucleotide

The two C-4 protons of reduced nicotinamide adenine dinucleotide (NADH) produce an AB NMR spectrum at 100 MHz as well as at 220 MHz. This observation allows an upper limit of 50 sec^?1 to be placed on the mean rate of interconversion of the two folded forms of NADH invoked to account for the magnetic non-equivalence of the C-4 protons. The interpretation of non-equivalence of the C-4 protons in terms of the various equilibria among folded and unfolded forms of NADH and its possible significance in the mechanism of action of dehydrogenase enzymes is discussed. It is suggested that one folded form of NADH is strongly favored thermodynamically over the other and that the resulting magnetic non-equivalence of the C-4 protons is of doubtful significance in explaining the stereospecificity of dehydrogenase enzymes toward the nicotinamide ring.     

烟酰胺辅酶在聚甲苯胺蓝膜修饰的玻碳电极上的电催化氧化

用循环伏安法在玻碳电极上电沉积一层稳定的甲苯胺蓝聚合物膜 ,研究了这层膜在 0 .2mol/L磷酸缓冲溶液 (pH 6 .86 )中的电化学性质 ,并且考察了该膜修饰的玻碳电极对烟酰胺辅酶 (NADH)的电催化作用 ,用旋转圆盘电极测量了NADH在该修饰电极上的催化反应常数。实验发现 ,在该修饰电极上 ,NADH氧化峰电位比未修饰的玻碳电极负移了 4 5 0mV ,且其催化反应速率常数为 3.5× 10 3 L·mol-1·s-1,说明聚甲苯胺蓝膜对NADH有良好的电催化作用     

高效毛细管电泳—电化学法检测烟酰胺腺嘌呤二核苷酸

报道了一种测定烟酰胺腺嘌呤二核苷酸的毛细管电泳电化学检测方法。采用３０ｃｍ×２５μｍ的石英毛细管分离ＮＡＤＨ与脲酸，以微型碳糊电极测定经分离后的ＮＡＤＨ的含量。在ｐＨ７．５的磷酸盐缓冲溶液中，标准工作曲线的范围为１．０－１００μｍｏｌ／Ｌ；最代检测浓度为０．６０μｍｏｌ／Ｌ。