Synthesis and characterization of oligonucleotide conjugates bearing electroactive labels

Oxime bond formation has been applied to the preparation of oligonucleotides labeled with electrochemical ferrocene and viologen labels. Aminooxy functionalized ferrocene and viologen derivatives were prepared by a straightforward route and efficiently conjugated with aldehyde containing oligonucleotides either at 3′ or 5′ end. Both labels were found to not disturb the recognition properties of the oligonucleotide. The versatility of the method was further demonstrated by preparing bi-functionalized conjugates with a disulfide at 3′ end and an electrochemical label at 5′ end.     

Aldehydic Oligonucleotide: A Key Intermediate for the Preparation of Oligonucleotide Conjugates Through Oxime Bond Formation

Oligonucleotides functionalized with an aldehyde group are the key intermediates used for the preparation of peptide-oligonucleotide conjugates through the formation of an oxime linkage. Herein, we describe a brief overview of various synthetic protocols developed in our laboratory for the preparation of aldehyde containing oligonucleotides and their subsequent conjugation with peptides.     

DNA or RNA oligonucleotide 2'-hydrazides for chemoselective click-type ligation with carbonyl compounds

An efficient method for the synthesis of DNA or RNA oligonucleotide 2'-hydrazides is described. Fully deprotected oligonucleotides containing a hydrazide group at the 2'-position of a uridine residue were obtained by a novel two-step procedure: periodate cleavage of an oligonucleotide with 1,2-diol group followed by conversion of the aldehyde to hydrazide with an extended linker arm using a homobifunctional reagent succinic dihydrazide and NaBH(3)CN. The resulting oligonucleotide 2'-hydrazides were efficiently conjugated by a click-type reaction at acidic pH to aliphatic or aromatic aldehydes with or without NaBH(3)CN reduction to afford novel 2'-conjugates.     

介质表面修饰对蛋白质芯片固定率和反应性的影响

评价最常用的二种玻璃表面修饰方法对蛋白质芯片质量的影响．选择蛋白质的固定效率、反应性作为检测指标,对戊二醛修饰法和多聚赖氨酸修饰法进行比较,由机械手将探针蛋白质分别固定在两种玻片上,靶蛋白用荧光染料Cy3标记,两种修饰方法的芯片均可使蛋白质保持较好的固定效率和反应活性．由共价键偶联的醛基修饰玻片制备的蛋白质芯片不仅有更高的反应活性,而且图象佳,但背景偏高、用醛基修饰的玻片制备蛋白质芯片是较理想的选择、     

An induced aliphatic aldehyde dehydrogenase from the bioluminescent bacterium, Beneckea harveyi. Purification and properties.

A NAD+-dependent aldehyde dehydrogenase, the activity of which induces at the same time as luceriferase, has been purified from the bioluminescent bacterium Beneckea harveyi, and its chemical and physical properties have been investigated. The purification is accomplished in three steps resulting in an enzyme preparation that gives a single protein band on three different gel electrophoresis systems. The molecular weight of the purified enzyme was estimated to be 120,000 by gel filtration. Sodium dodecyl sulfate-gel electrophoresis gave a molecular weight of 59,000 indicating that aldehyde dehydrogenase has a dimeric structure with subunits of similar molecular weight. The purified enzyme has a high specificity for long chain aliphatic aldehydes; the Michaelis constants for aldehydes decrease with increasing chain length as also observed for bacterial aldehyde dehydrogenases involved in the metabolism of hydrocarbons. The aldehyde specificity of the aldehyde dehydrogenase is similar to that of luciferase indicating that the functional role of the enzyme may be linked with the bioluminescent system.     

Measurement of the formation of betaine aldehyde and betaine in rat liver mitochondria by a high pressure liquid chromatography-radioenzymatic assay.

A new assay procedure for measurement of rat liver mitochondrial choline dehydrogenase was developed. Oxidation of [methyl-14C]choline to [methyl-14C]betaine aldehyde and [methyl-14C]betaine was measured after isolating these compounds using HPLC. We observed that NAD+ was required for conversion of betaine aldehyde to betaine in rat liver mitochondria. In the absence of this cofactor, oxidation of choline led to the accumulation of betaine aldehyde. The apparent Km of the mitochondrial choline dehydrogenase for choline was 0.14-0.27 mM, which is significantly lower than previously reported. A partially purified preparation of choline dehydrogenase catalyzed betaine aldehyde formation only in the presence of exogenous electron acceptors (e.g., phenazine methosulfate). This preparation failed to catalyze the formation of betaine even in the presence of NAD+, indicating that betaine aldehyde dehydrogenase may be a separate enzyme from choline dehydrogenase.     

Biogenic Aldehydes in Brain: On Their Preparation and Reactions with Rat Brain Tissue

When 1 mM serotonin, dopamine, or norepinephrine was incubated with a monoamine oxidase preparation (mitochondrial membranes) in the presence of 4 mM sodium bisulfite, 85-95% of the amines were oxidized to the corresponding aldehydes. In the absence of bisulfite, the recoveries were only approximately 30%, and dark colored products were formed during the incubations. The aldehydes derived from tyramine, octopamine, methoxytyramine, and normetanephrine were also prepared by the use of this method. The bisulfite-aldehyde compounds were stable during storage at -20 degrees C. Bisulfite-free aldehyde solutions were made by diethylether extraction. When the aldehydes derived from dopamine or serotonin were incubated with rat brain homogenates, they were found to disappear in an aldehyde dehydrogenase- and aldehyde reductase-independent manner. The disappearance of the latter aldehyde was more pronounced, and the results indicated that this aldehyde may react with both proteins and phospholipids.     

Assay of brain aldehyde dehydrogenase activity using high-performance liquid chromatography with electrochemical detection

A method has been developed for assay of aldehyde dehydrogenase (ALDH) in brain tissue or in other tissues containing low ALDH-activity. The aldehyde of dopamine was used as the substrate, and the 3,4-dihydroxyphenylacetic acid formed was measured using high-performance liquid chromatography (HPLC) with electrochemical detection. The aldehyde was prepared enzymatically by incubating dopamine with a monoamine-oxidase preparation from rat liver mitochondria in the presence of Na+-bisulfite in 10 mM K+-phosphate buffer (pH 7.5). Rat brain homogenates were incubated in 50 mM Na+-pyrophosphate buffer (pH 8.8) containing 0.5 mM NAD+ and 5 microM aldehyde. The reaction was terminated with perchloric acid containing Na+-bisulfite to trap excess of the aldehyde. The acid supernatants were injected on a reverse-phase HPLC column and elution was performed with citrate buffer, pH 2.50. The method permits assay with 1-10 mg of brain tissue with an overall precision of 3%. The assay rate was 5-6 samples per hour.     

Solid phase synthesis of peptidyl aldehydes from C-terminal thiazolidinyl peptides

Summary Peptidyl aldehydes are potent transition state analogue inhibitors of cysteine and serine proteinases. The aldehyde function has recently been used for chemoselective peptide ligation. The preparation of peptidyl aldehydes on a solid support requires that the aldehyde be masked during peptide elongation and generated in a final step under mild conditions. We report here the preparation of peptidyl aldehydes by copper salt-mediated neutral hydrolysis of the corresponding C-terminal thiazolidinyl peptides which were elongated on a solid support.     

Two efficient polymeric chemical platforms for oligonucleotide microarray preparation

In this report we describe two robust procedures for oligonucleotide microarray preparation based on polymeric coatings. The proposed chemical approaches include: 1) a glass functionalisation step with appropriate silanes (gamma-aminopropyltriethoxysilane-APTES or 3-glycidoxypropyltrimethoxysilane-GOPS), 2) a coating step using polymers (poly-L-Lysine or poly(acrylic acid-co-acrylamide) copolymer) covalently bound to the modified glass and 3) a surface activation step to allow for the attachment of amino-modified oligonucleotides. Results obtained using these chemistries in oligo microarray preparation show: 1) an overall high loading capacity and availability to hybridisation against targets, 2) a good uniformity, 3) resistance to consecutive probing/ stripping cycles, 4) stability to thermal cycles, 5) effectiveness in hybridisation-mediated mutation detection procedures and 6) the possibility to perform enzymatic reactions, such as ligation.     

NAD-linked, GSH- and factor-independent aldehyde dehydrogenase of the methylotrophic bacterium, Hyphomicrobium X

Cell-free extracts of Hyphomicrobium X showed NAD-dependent aldehyde dehydrogenase activity, provided that NAD addition preceded that of aldehyde. Activity was lost rather rapidly, especially during purification attempts, but this could be partially masked by including a time-dependent restoration step with thiol compounds in the protocol. The nature of the assay buffer appeared to be critical and stimulation occurred on incorporation of K+ ions in the mixture. An even higher specific activity could be achieved by 1,4-dithiothreitol (DTT) treatment of the preparation, followed by removal of DTT, and assaying in the absence of thiol compounds under anaerobic conditions. Exposure of such a preparation to O2 led to a significant decrease in activity within a couple of hours. Immediate inactivation occurred on addition of H2O2, but this could be prevented completely by prior addition of NAD. Since GSH does not participate in the reaction and no stimulating factor was detected, the role of thiol compounds is most probably confined to restoration or prevention of damage to an O2-sensitive, necessary thiol group. Since the same features were found for cell-free extract as for the partially purified enzyme, only one enzyme type seems to be present. Although the enzyme is a general aldehyde dehydrogenase, the kinetic parameters and the specific activity of the cell-free extract for formaldehyde indicate that it may play a role in formaldehyde dissimilation by Hyphomicrobium X. The NAD-linked, GSH- and factor-independent aldehyde dehydrogenase described here appears to be different in several respects from the formaldehyde dehydrogenase of Pseudomonas putida (EC 1.2.1.46) (despite showing similar behavior toward coenzymes and factors) but resembles the aldehyde dehydrogenase from baker's yeast (EC 1.2.1.5).     

Rapid separation of dehydrogenases by affinity chromatography with new induced specificity phases

We describe a procedure using immobilized nicotinamide as an affinity chromatographic ligand for the binding of NAD(P)+-dependent dehydrogenases. The procedure involves preparation of nicotinamide N1-(N-(6-aminohexyl)-acetamide)-agarose and modification of the immobilized nicotinamide by the addition of a ketone or an aldehyde to form an adduct. The nicotinamide, which has no affinity for dehydrogenase, becomes a very specific ligand of dehydrogenase, which binds the ketone or the aldehyde as substrate or inhibitor. In tests, the adduct prepared with immobilized nicotinamide and sodium pyruvate bound specifically to lactate dehydrogenase (EC 1.1.1.27), whereas the adduct prepared with alpha-ketoglutarate bound to glutamate dehydrogenase (EC 1.4.1.3). This technique enables the rapid isolation of a given dehydrogenase.     

Mechanism of action of uridine diphoglucose dehydrogenase. Evidence for an essential lysine residue at the active site

The oxidation of UDP-glucose by the enzyme UDP-glucose dehydrogenase (EC 1.1.1.22) from beef liver has been shown to proceed via the enzyme-bound intermediate, UDP-alpha-D-glyco-hexodialdose. The enzyme does not release this aldehyde, nor can it be trapped by reaction with hydroxylamine, thiosemicarbazide, or cyanide. Tight binding of the intermediate aldehyde can be explained by the recent observation that the essential thiol group of the enzyme forms a thiohemiacetal with the aldehyde during the course of the reaction. However, an enzyme preparation with the essential thiol derivatized with cyanide will still not release the aldehyde, indicating an additional as yet unknown binding mechanism. Derivatization ([14C]formaldehyde, followed by NaBH4 reduction) of 6 of the approximately 168 lysine residues per enzyme molecule (of six catalytic subunits) results in destruction of 47% of the enzyme activity, suggesting the involvement of an essential reactive lysine in the mechanism. Preincubation of the enzyme with UDP-glucose decreases both the loss of activity and incorporation of the label, indicating that this lysine is in the vicinity of the active site. Acid hydrolysis of the labeled preparation, followed by paper chromatography, shows that the label has a mobility, in the system used, that is identical with lysine. Elution of this spot followed by chromatography on Aminex A-5 resin showed that it contained the expected mixture of epsilon-N-methyl lysines. When enzyme that has its essential thiol derivatized with cyanide is incubated with UDP-[14C]glucose and NAD+, and then reduced with NaB3H4, a stable enzyme complex is formed which contains both labels. Acid hydrolysis of this preparation, followed by either two-dimensional paper chromatography or separation in an amino acid analyzer, results in both labels appearing in the position of lysine. It is evident that the enzyme oxidizes the UDP-[14C]glucose to the corresponding aldehyde which occurs as the Schiff's base with an essential lysine. This is then reduced by the NaB3H4 to form a secondary amine which is stable toward hydrolysis and migrates with lysine in separation procedures. As would be predicted, the enzyme can be similarly labeled by treatment with UDP-alpha-D-gluco-hexodisidose alone, followed by NaB3H4 reduction. The same hydrolysis product results from this procedure, and it behaves identically with the product formed by treating alpha-N-acetyl lysine with UDP-alpha-D-gluco-hexodialdose, reducing with NaBH4, and then hydrolyzing. This substance appears to be N5-((5-formyl-2-furanyl)methyl)lysine. When chromatographed on Aminex A-5, both the model compound and enzyme hydrolysate gave peaks corresponding to free lysine and the proposed derivative. Evidence is presented that the oxidation of UDP-glucose to the aldehyde is a concerted reaction involving the formation of the Schiff's base, rather than the formation of the aldehyde with the subsequent formation of the Schiff's base...     

Purification and characterization of choline oxidase from Arthrobacter globiformis

Choline oxidase was purified from the cells of Arthrobacter globiformis by fractionations with acetone and ammonium sulfate, and column chromatographies on DEAE-cellulose and on Sephadex G-200. The purified enzyme preparation appeared homogeneous on disc gel electrophoresis. The enzyme was a flavoprotein having a molecular weight of approx. 83,000 (gel filtration) or approx. 71,000 (sodium dodecyl sulfate--polyacrylamide disc gel electrophoresis) and an isoelectric point (pI) around pH 4.5. Identification of the reaction products showed that the enzyme catalyzed the following reactions: choline + O2 leads to betaine aldehyde + H2O2, betaine aldehyde + O2 + H2O leads to betaine + H2O2. The enzyme was highly specific for choline and betaine aldehyde (relative reaction velocities: choline, 100%; betaine aldehyde, 46%; N,N-dimethylaminoethanol, 5.2%; triethanolamine, 2.6%; diethanolamine, 0.8%; monoethanolamine, N-methylaminoethanol, methanol, ethanol, propanol, formaldehyde, acetaldehyde, and propionaldehyde, 0%). Its Km values were 1.2 mM for choline and 8.7 mM for betaine aldehyde. The optimum pH for the enzymic reaction was around pH 7.5.     

Alpha-oxo semicarbazone peptide or oligodeoxynucleotide microarrays

We describe in this paper the preparation and characterization of semicarbazide glass slides and their use for the fabrication of microarrays using site-specific alpha-oxo semicarbazone ligation. The functional density and homogeneity of the semicarbazide glass slides were optimized by analyzing the reactivity of the layer toward a synthetic glyoxylyl fluorescent probe. Oligonucleotide microarrays were prepared by site-specific immobilization of glyoxylyl oligodeoxynucleotides. The slides were directly used in the hybridization assays using fluorescence detection and displayed a significant gain in sensibility as compared to the aldehyde glass slide/amino oligodeoxynucleotide chemistry. Semicarbazide slides were also used for the immobilization of a biotinylated peptide alpha-oxo aldehyde. The peptide microarrays allowed model interaction studies with streptavidin or an anti-biotin antibody.     

Reproducible and inexpensive probe preparation for oligonucleotide arrays

We present a new protocol for the preparation of nucleic acids for microarray hybridization. DNA is fragmented quantitatively and reproducibly by using a hydroxyl radical-based reaction, which is initiated by hydrogen peroxide, iron(II)-EDTA and ascorbic acid. Following fragmentation, the nucleic acid fragments are densely biotinylated using a biotinylated psoralen analog plus UVA light and hybridized on microarrays. This non-enzymatic protocol circumvents several practical difficulties associated with DNA preparation for microarrays: the lack of reproducible fragmentation patterns associated with enzymatic methods; the large amount of labeled nucleic acids required by some array designs, which is often combined with a limited amount of starting material; and the high cost associated with currently used biotinylation methods. The method is applicable to any form of nucleic acid, but is particularly useful when applying double-stranded DNA on oligonucleotide arrays. Validation of this protocol is demonstrated by hybridizing PCR products with oligonucleotide-coated microspheres and PCR amplified cDNA with Affymetrix Cancer GeneChip microarrays.     

In situ synthesis of oligonucleotide microarrays.

This contribution presents a brief overall look of the methods for the preparation of various types of DNA microarrays and a thorough examination of the methods for in situ synthesis of oligonucleotide microarrays.     

Disulfiram inhibition of aldehyde reductase from the rat liver

Disulphiram (tetraethylthiuram disulphide teturam, antabus), the known antialcoholic preparation, is studied for its effect on the aldehyde reductase activity (EC 1.1.1.1) in the rats' liver. Apparent Km and V are calculated for acetylaldehyde and NADH as well as Ki of disulphiram relative to the substrate and cofactor of the enzyme. The obtained data permit considering disulphiram a high-specific inhibitor of aldehyde reductase in rats' liver.