Synthesis, DNA polymerase incorporation, and enzymatic phosphate hydrolysis of formamidopyrimidine nucleoside triphosphates

The nucleoside triphosphates of N6-(2-deoxy-alpha,beta-d-erythro-pentofuranosyl)-2,6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy.dGTP) and its C-nucleoside analogue (beta-C-Fapy.dGTP) were synthesized. The lability of the formamide group required that nucleoside triphosphate formation be carried out using an umpolung strategy in which pyrophosphate was activated toward nucleophilic attack. The Klenow fragment of DNA polymerase I from Escherichia coli accepted Fapy.dGTP and beta-C-Fapy.dGTP as substrates much less efficiently than it did dGTP. Subsequent extension of a primer containing either modified nucleotide was less affected compared to when the native nucleotide is present at the 3'-terminus. The specificity constants are sufficiently large that nucleoside triphosphate incorporation could account for the level of Fapy.dG observed in cells if 1% of the dGTP pool is converted to Fapy.dGTP. Similarly, polymerase-mediated introduction of beta-C-Fapy.dG could be useful for incorporating useful amounts of this nonhydrolyzable analogue for use as an inhibitor of base excision repair. The kinetic viability of these processes is enhanced by inefficient hydrolysis of Fapy.dGTP and beta-C-Fapy.dGTP by MutT, the E. coli enzyme that releases pyrophosphate and the corresponding nucleoside monophosphate upon reaction with structurally related nucleoside triphosphates.     

Cross-coupling reactions of nucleoside triphosphates followed by polymerase incorporation. Construction and applications of base-functionalized nucleic acids

Construction of functionalized nucleic acids (DNA or RNA) via polymerase incorporation of modified nucleoside triphosphates is reviewed and selected applications of the modified nucleic acids are highlighted. The classical multistep approach for the synthesis of modified NTPs by triphosphorylation of modified nucleosides is compared to the novel approach consisting of direct aqueous cross-coupling reactions of unprotected halogenated nucleoside triphosphates. The combination of cross-coupling of NTPs with polymerase incorporation gives an efficient and straightforward two-step synthesis of modified nucleic acids. Primer extension using biotinylated templates followed by separation using streptavidine-coated magnetic beads and DNA duplex denaturation is used for preparation of modified single stranded oligonucleotides. Examples of using this approach for electrochemical DNA labelling and bioanalytical applications are given.     

Expansion of repertoire of modified DNAs prepared by PCR using KOD Dash DNA polymerase

Thymidine analogues bearing a variety of functional groups at the C5-position via an amino-linker arm were prepared and the substrate activity for PCR using thermophilic KOD Dash DNA polymerase was examined. The enzyme accepted the thymidine analogues bearing pyridine, imidazole, biotin, a cationic-charged guanidinium, a cationic-charged amino, mercaptopyridyl and phenanthrolne groups at the C5-position, forming the corresponding PCR product. However, a thymidine analogue bearing a carboxyl group at the C5-position was a poor substrate and the corresponding PCR products could not be obtained. The thymidine analogue bearing a mercapto group was also a poor substrate for the enzyme, because it dimerized by disulfide linkage under PCR conditions. The enzyme hardly accepts the thymidine analogues with a negatively-charged carboxyl group or a bulky group as a substrate. KOD Dash DNA polymerase, having a broader substrate specificity than any other DNA polymerase, will expand the variety of modified DNAs that can be prepared by PCR.     

Elucidation of the initial step of oligonucleotide fragmentation in matrix-assisted laser desorption/ionization using modified nucleic acids

To probe the mechanism of gas-phase oligonucleotide ion fragmentation, modified oligonucleotides were studied using matrix-assisted laser desorption/ionization. The oligonucleotides were of the form 5'-TTTTXTTTTT, where X was a modified nucleotide. Modifications included substitution of hydroxy, methoxy, amino, and allyl groups at the 2'-position of the deoxyribose. The modified ribose contained adenine, guanine, cytosine, or uracil bases. For comparison, we studied oligomers where X was an unmodified adenosine, guanosine, cytidine, thymidine, or uridine deoxyribonucleotide. We found a very strong dependence of the matrix-to-analyte ratio on fragmentation for these oligomers. Analysis of these modifications suggests that the initial fragmentation step in MALDI-MS involves a two-step (E1) elimination of the base.     

Development of a novel nucleoside analogue with S-type sugar conformation: 2′-deoxy-trans-3′,4′-bridged nucleic acids

Two novel trans-3′,4′-bridged nucleic acid (trans-3′,4′-BNA) monomers, one with a 3,5,8-trioxabicyclo[5.3.0]decane structure and the other with a 4,7-dioxabicyclo[4.3.0]nonane structure, were successfully synthesized from thymidine. The locked trans-fused ring structures of the nucleoside analogues were confirmed by X-ray crystallography, which also indicated that their furanose rings had a typical S-type conformation involving C_2′-endo or C_3′-exo sugar puckering, respectively, and the same ring conformation as that observed in the B-type helical structure of the DNA duplex.     

DNA/羟基磷灰石杂化膜修饰电极用于研究DNA与量子点的相互作用

首次利用溶胶-凝胶一步法制备了纳米多孔羟基磷灰石(HAp)-DNA杂化膜修饰玻碳电极,HAp优良的生物相容性和独特的吸附性可以将DNA固定在HAp多孔薄膜上,而DNA的大分子结构对HAp膜起到稳定剂的作用.采用循环伏安法和交流阻抗法系统地研究了电极表面固定DNA的稳定性以及固定的DNA与非电活性核壳型量子点CdTe/C...     

Electrochemical detection of hybridization using peptide nucleic acids and methylene blue on self-assembled alkanethiol monolayer modified gold electrodes

An electrochemical hybridization biosensor based on peptide nucleic acid (PNA) probes is presented. PNA probes were attached covalently through a competition of free amines on the guanine bases and also at the 5^′ end of the probe, using N-(3-dimethylamino)propyl)-N^′-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) onto a carboxylate terminated alkanethiol self-assembled monolayer (SAM) preformed on a gold electrode (AuE). The covalently immobilized probe could selectively hybridize with the target DNA to form a hybrid on the surface despite the bases being attached to the SAM. The changes in the peak currents of methylene blue (3,7-bis(dimethylamino)phenothiazin-5-ium chloride, MB), an electroactive label, were observed upon hybridization of probe with the target. Effective discrimination against point mutation was also obtained. Performance characteristics of the sensor are described, along with future prospects.     

DNA circuits as amplifiers for the detection of nucleic acids on a paperfluidic platform

This article describes the use of non-enzymatic nucleic acid circuits based on strand exchange reactions to detect target sequences on a paperfluidic platform. The DNA circuits that were implemented include a non-enzymatic amplifier and transduction to a fluorescent reporter; these yield an order of magnitude improvement in detection of an input nucleic acid signal. To further improve signal amplification and detection, we integrated the enzyme-free amplifier with loop-mediated isothermal amplification (LAMP). By bridging the gap between the low concentrations of LAMP amplicons and the limits of fluorescence detection, the non-enzymatic amplifier allowed us to detect as few as 1200 input templates in a paperfluidic format.     

Study on preparation of modified lubricant containing nano-Schiff base and Schiff base copper complex in W/O microemulsion reactor

A successful preparation of Schiff base and Schiff base copper complex was carried out directly in polar base oil (vegetable oil) using a water/oil microemulsion reactor. The prepared nanometer sized Schiff base and Schiff base copper complex dispersed uniformly and spontaneously in the oil. The nanometer sized particles of the Cu(II) chelate of bissalicylaldehyde-ethylenediamine and the bissalicylaldehyde-ethylenediamine in oil were observed directly by SEM. Owing to a modification of the polar base oil (vegetable oil) by 1 wt % of nano-scale Cu(II) chelate of bissalicylaldehyde-ethylenediamine and 1 wt % of bissalicylaldehyde-ethylenediamine, the last nonseizure load had gone up 40% over that of the original ones. It was verified by AES analysis that steel/steel rubbing pairs went through a selective transfer process under lubrication with the modified polar lubricant. It was suggested that the mechanism of the improvement of tribological characteristics of the modified lubricant was selective transfer effect. An antibacterial activity of the modified lubricant was inspected also. The article is published in the original.     

Electrochemical biosensor for detection of BCR/ABL fusion gene based on hairpin locked nucleic acids probe

This communication reports on a novel biosensor to study the hybridization specificity by using thiolated hairpin locked nucleic acids (LNA) as the capture probe. The LNA probe was immobilized on the gold electrode through sulfur–Au interaction and could selectively hybridize with its target DNA. Differential pulse voltammetry (DPV) was used to monitor the hybridization reaction on the probe electrode. The decrease of the peak current of methylene blue, an electroactive indicator, was observed upon hybridization of the probe with the target DNA. The results indicated this new method has excellent specificity for single-base mismatch and complementary after hybridization, and a high sensitivity. This LNA probe has been used for assay of fusion gene in Chronic Myelogenous Leukemia (CML) of the real sample with satisfactory result.     

The trapping of a spontaneously "flipped-out" base from double helical nucleic acids by host-guest complexation with beta-cyclodextrin: the intrinsic base-flipping rate constant for DNA and RNA

Beta-cyclodextrin, which forms stable host-guest complexes with purine bases, induces the melting of RNA and DNA duplexes below their normal melting temperatures. Alpha-cyclodextrin, which does not form stable complexes, has no effect on either RNA or DNA. Gamma-cyclodextrin, which forms weaker complexes, has no effect on RNA and a smaller effect than beta-cyclodextrin on DNA. The rate of melting is kinetically first-order in duplex and, above about 20 mM beta-cyclodextrin, is independent of the beta-cyclodextrin concentration with a first-order rate constant, common to both RNA and DNA, of (3.5 +/- 0.5) x 10(-3) s(-1) at 61 degrees C (DNA) and at 50 degrees C (RNA). This is taken to be the rate constant for spontaneous "flipping out" of a base from within the duplex structure of the nucleic acids, the exposed base being rapidly trapped by beta-cyclodextrin. Like beta-cyclodextrin, nucleic acid methyltransferases bind the target base for methylation in a site that requires it to have flipped out of its normal position in the duplex. The spontaneous flip-out rate constant of around 10(-3) s(-1) is near the value of k(cat) for the methyltransferases (ca. 10(-3) to 10(-1) s(-1)). In principle, the enzymes, therefore, need effect little or no catalysis of the flipping-out reaction. Nevertheless, the flip-out rate in enzyme/DNA complexes is much faster. This observation suggests that the in vivo circumstances may differ from in vitro models or that factors other than a simple drive toward higher catalytic power have been influential in the evolution of these enzymes.     

Study of the suitability of silica based xerogels synthesized using ethyltrimethoxysilane and/or methyltrimethoxysilane precursors for aerospace applications

Silica based materials were synthesized using ethyltrimethoxysilane (ETMS) and/or methyltrimethoxysilane (MTMS) precursors by sol–gel technology, in order to ascertain if their properties are suitable for aerospace applications. When ETMS was used alone and in equimolar ETMS/MTMS mixtures, no gel formation took place and a resin-like precipitate was observed. After drying, a compact tablet was formed. When mixtures of 25% ETMS/75% MTMS and MTMS alone were used, gel formation occurred and xerogels were produced upon drying. Chemical and structural characterization of the obtained materials was performed using Elemental Analysis, FTIR, XRD and SEM. Bulk density, specific surface area, contact angle and the thermal behavior were also evaluated. For materials from ETMS, the chemical structure grows preferentially in one direction and, in the case of MTMS the growth follows a 3D pattern. The use of ETMS precursor leads to a significant increase in the product density, accompanied by a decrease in the specific surface area. It also leads to a decrease in the thermal stability limit of the synthesized materials. Then, ETMS precursor is less appropriate than MTMS precursor for space applications. However, ETMS co-precursor in mixtures with MTMS contributes to the increase in the hydrophobic character of the synthesized materials.     

Cyclohexanyl peptide nucleic acids (chPNAs) for preferential RNA binding: effective tuning of dihedral angle beta in PNAs for DNA/RNA discrimination

[structures: see text] A serious drawback of peptide nucleic acids (PNAs) from an application perspective that has not been adequately dealt with is nondiscrimination of identical DNA and RNA sequences. An analysis of the available X-ray and NMR solution structures of PNA complexes with DNA and RNA suggested that it might be possible to rationally impart DNA/RNA duplex binding selectivity by tuning the dihedral angle beta of the flexible ethylenediamine part of the PNA backbone (II) via suitable chemical modifications. Cyclohexanyl PNAs (chPNAs) with beta approximately = 65 degrees were designed on the basis of this rationale. The chPNAs introduced remarkable differences in duplex stabilities among their DNA and RNA complexes, with melting temperatures (deltaTm(RNA-DNA) = +16-50 degrees C) depending on the number of modifications and the stereochemistry. This is a highly significant, exceptional binding selectivity of a mix sequence of PNA to RNA over the same DNA sequence as that seen to date. In contrast, cyclopentanyl PNAs (cpPNAs) with beta approximately = 25 degrees hybridize to DNA/RNA strongly without discrimination because of the ring puckering of the cyclopentane ring. The high affinity of chPNAs to bind to RNA without losing base specificity will have immediate implications in designing improved PNAs for therapeutic and diagnostic applications.     

Preliminary studies on the processing sequence for southern red oak and municipal solid waste using a hybrid dilute acid/ enzymatic hydrolysis process for ethanol production

Based on this preliminary study, a metric ton of dry southern red oak chips subjected to a first-stage dilute sulfuric acid hydrolysis would yield 132 kg of xylose and 40 kg of glucose and mannose. A second-stage dilute sulfuric acid hydrolysis on the first-stage residue would yield only 128 kg of additional glucose, but a second-stage cellulytic enzyme hydrolysis on the first-stage residue would yield an additional 265 kg of glucose. Fermentation of these hydrolyzates would show that the hybrid process would yield over 50% more ethanol. Results on other biomass are also included.     

Quantification of the 35S promoter in DNA extracts from genetically modified organisms using real-time polymerase chain reaction and specificity assessment on various genetically modified organisms, part I: operating procedure

A highly sensitive quantitative real-time assay targeted on the 35S promoter of a commercial genetically modified organism (GMO) was characterized (sF/sR primers) and developed for an ABI Prism 7700 Sequence Detection System and TaqMan chemistry. The specificity assessment and performance criteria of sF/sR assay were compared to other P35S-targeted published assays. sF/sR primers amplified a 79 base pair DNA sequence located in a part of P35S that is highly conserved among many caulimovirus strains, i.e., this consensus part of CaMV P35S is likely to be present in many GM events. According to the experimental conditions, the absolute limit of detection for Bt176 corn was estimated between 0.2 and 2 copies of equivalent genome (CEG). The limit of quantification was reached below 0.1% Bt176 content. A Cauliflower Mosaic Virus control (CaMV) qualitative assay targeted on the ORF III of the viral genome was also used as a control (primers 3F/3R) to assess the presence of CaMV in plant-derived products. The specificity of this test was assessed on various CaMV strains, including the Figwort Mosaic Virus (FMV) and solanaceous CaMV strains. Considering the performance of sF/sR quantification test, the highly conserved sequence, and the small size of the amplicon, this assay was tested in a collaborative study in order to be proposed as an international standard.     

Ultrasensitive amperometric determination of PSA based on a signal amplification strategy using nanoflowers composed of single-strand DNA modified fullerene and Methylene Blue,and an improved surface-initiated enzymatic polymerization

The authors describe a signal amplification strategy for highly sensitive detection of the prostate-specific antigen (PSA). This is accomplished by a combination of two methods, viz. (a) improved surface-initiated enzymatic polymerization (SIEP), and (b) the use of nanoflowers prepared from C₆₀ fullerene and Methylene Blue (C₆₀/MB) modified with a long single-strand DNA. C₆₀/MB acts as a novel electrochemical indicator. The C₆₀/MB nanoflowers improve the load of MB and promote the electron transfer. The integration of the SIEP technique and the C₆₀/MB nanomaterial also results in improved loading of MB on the nucleic acid. Ultimately, dual cascade signal amplification is accomplished. The biosensor was constructed as follows: (a) Gold nanospheres were modified with antibody 2 (Ab₂) and a thiolated oligonucleotide (referred to as S₀). (2) S₀ is then extended by the SIEP reaction. (3) The redox indicator C₆₀/MB is then connected to the extended guanine-rich ssDNA which then yields the amperometric signal. (4) A sandwich immunoassay is performed by capturing the nanoprobe oy type Ab₂-Au-S₀ on the gold electrode modified with multi-walled carbon nanotubes (MWCNTs) and protein A. Current is measured by using differential pulse voltammetry (DPV). The synergic effect of the biofunctional nanomaterial and the signal amplification strategy greatly improves the performance of this immunoassay. Under optimized conditions and at a working voltage of typically ?0.18 V (vs Ag/AgCl), the assay has a linear range that extends from 15 pg·mL^?1 to 8 ng·mL^?1 of PSA. The detection limit is as low as 1.7 pg·mL^?1 (at an S/N ratio of 3). In our perception, this dual amplification scheme has a wide scope in that it may become applicable to numerous other immunoassays.

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Graphical abstract C₆₀/Methylene blue nanoflowers, a novel electrochemical indicator, connect with the long single-stranded DNA (ssDNA) extended by the improved surface-initiated enzymatic polymerization method. This amplification strategy is utilized to construct a sandwich prostate-specific antigen (PSA) immunosensor.

     

Development,validation and application of a 96-well enzymatic assay based on LC-ESI-MS/MS quantification for the screening of selective inhibitors against Mycobacterium tuberculosis purine nucleoside phosphorylase

Mycobacterium tuberculosis (Mtb) purine nucleoside phosphorylase (PNP, EC 2.4.2.1) has been identified as a target for the development of specific inhibitors with potential antimycobacterial activity. We hereby described the development and validation of a new 96-well LC-ESI-MS/MS method to assess the inhibition activity of nucleoside analogues towards MtbPNP and the human PNP (HsPNP). Enzyme activity was determined by monitoring the phosphorolysis of inosine (Ino) to hypoxanthine (Hpx). The enzymatic assay (v = 0.5 mL, enzyme<0.2 μg/well, T = 37 °C) was performed with an overall time of about 15 min/plate for sample processing and 2 min/sample for LC-MS analysis. Validation of the quantification method met the criteria of the CDER guidance of FDA. Kinetic parameters were in agreement with those reported in literature (HsPNP K_M = 0.150 ± 0.020 mM vs 0.133 ± 0.015 mM; MtbPNP K_M = 0.060 ± 0.009 mM vs 0.040 ± 0.003 mM for Ino), thus demonstrating the reliability of the newly developed enzymatic assay. Preliminary inhibition assays confirmed the effects reported for Acyclovir (Acv) and Formycin A (FA) against HsPNP and MtbPNP. The validated enzymatic assay was applied to the evaluation of a set of 8-halo-, 8-amino-, 8-O-alkyl-substituted purine ribonucleosides synthesized on purpose as potential inhibitors against MtbPNP. The assayed 8-substituted ribonucleosides did not exert a significant inhibitory effect against the tested enzymes up to 1 mM.     

A kinetic fluorometric method for the determination of nucleic acids using a ternary equilibrium system of nucleic acids—iron (III) tetracarboxy phthalocyanine-poly-lysine coupled with the oxidation reaction between hydrogen peroxide and dl-tyrosine

Using the oxidation reaction between hydrogen peroxide and dl-tyrosine as fluorescence indication, the evident tuning effect of nucleic acids on catalytic activity of mimetic enzyme iron (III) tetracarboxy phthalocyanine (FeC₄Pc) in the presence of poly-lysine was observed and studied. The oxidation reaction between hydrogen peroxide and dl-tyrosine with FeC₄Pc as catalyst gave an intensively fluorescent compound, which has an excitation wavelength of 325 nm and an emission wavelength of 418 nm. The fluorescence was quenched by a proper concentration of poly-lysine due to its association with FeC₄Pc and consequently the descent of the catalytic activity of FeC₄Pc, but recovered by addition of nucleic acids. Under optimal conditions, the recovered fluorescence is proportional to the concentration of nucleic acids. Based on the fact, a kinetic fluorescent method was developed for the determination of nucleic acids. The calibration graphs are linear over the range 10-2000 ng/mL both for fish sperm DNA (FS DNA) and calf thymus DNA (CT DNA). The corresponding detection limits are 1.04 ng/mL for FS DNA and 1.18 ng/mL for CT DNA, respectively. Four synthetic and three real nucleic acid samples were determined with satisfactory results.