Single-stranded RNA as primers of terminal deoxynucleotidyl transferase for template-independent DNA polymerization

Terminal deoxynucleotidyl transferase(Td T) has been characterized as template-independent polymerase using single-stranded DNA(ss DNA) as primers to generate random oligonucleotides. However, the extension performance of Td T to single-stranded RNA(ss RNA) is vague. By systematically comparing and contrasting the performance of Td T-catalyzed ss DNA and ss RNA extension, it is indicated that the catalytic efficiency of ss RNA as primers was about 3 times lower than ss DNA as primers. Collective...     

Fluorometric determination of microRNA via FRET between silver nanoclusters and CdTe quantum dots

This paper describes a CdTe quantum dot-based fluorescence resonance energy transfer (FRET) based assay for the detection of the breast cancer biomarker microRNA. The method relies on energy transfer between DNA-templated silver nanoclusters (AgNCs) and CdTe QDs. Interaction between double strand oligonucleotide and QDs can be detected qualitatively through gel analysis and quantitatively by the signal amplification from AgNCs to QDs via FRET, best measured at an excitation wavelength of 350 nm and at emission wavelengths of 550 and 590 nm. Three microRNAs (microRNA-21, microRNA-155 and Let-7a) were quantified to verify the feasibility of the method, and a high sensitivity for microRNAs was achieved. Fluorescence intensity increases linearly with the log of the concentration of microRNA 155 in the 5.0 pM to 50 nM range, with a 1.2 pM detection limit.

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Graphical abstract Schematic presentation of a quantum dot-based (QD-based) fluorescence resonance energy transfer technique for the detection of microRNA (miRNA). The method relies on energy transfer between DNA-templated silver nanoclusters (AgNCs) and QDs.

     

Tail-labelling of DNA probes using modified deoxynucleotide triphosphates and terminal deoxynucleotidyl transferase. Application in electrochemical DNA hybridization and protein-DNA binding assays

A simple approach to DNA tail-labelling using terminal deoxynucleotidyl transferase and modified deoxynucleoside triphosphates is presented. Amino- and nitrophenyl-modified dNTPs were found to be good substrates for this enzyme giving 3'-end stretches of different lengths depending on the nucleotide and concentration. 3-Nitrophenyl-7-deazaG was selected as the most useful label because its dNTP was efficiently incorporated by the transferase to form long tail-labels at any oligonucleotide. Accumulation of many nitrophenyl tags per oligonucleotide resulted in a considerable enhancement of voltammetric signals due to the nitro group reduction, thus improving the sensitivity of electrochemical detection of the tail-labelled probes. We demonstrate a perfect discrimination between complementary and non-complementary target DNAs sequences by tail-labelled hybridization probes as well as the ability of tumour suppressor p53 protein to recognize a specific binding site within tail-labelled DNA substrates, making the methodology useful in electrochemical DNA hybridization and DNA-protein interaction assays.     

Detection of biotin-streptavidin interactions via terminal protection of small molecule linked DNA and the formation of fluorescent DNA-templated silver nanoclusters

It is known that the binding of certain proteins to small molecules in ssDNA/small-molecule chimeras protects the conjugated ssDNA from degradation by exonuclease I (Exo I). This has resulted in numerous methods to specifically detect the interaction between small molecules and proteins. We are presenting here an approach that utilizes the terminal protection strategy in combination with the formation of ssDNA-templated silver nanoclusters (AgNCs), thereby providing a fluorometric tool for the detection of such interactions. A C-rich ssDNA (type 5′-CCCCACCCCT-3′) was labelled with biotin at the 3′ end. In the absence of streptavidin (SA), the biotinylated ssDNA is hydrolyzed in the 3′ to 5′ direction by Exo I to form mononucleotides. The formation of the AgNCs is prevented due to the lack of the DNA scaffold, and this results in weak fluorescence. Conversely, in the presence of SA, the specific binding of SA to the biotinylated ssDNA protects the ssDNA from digestion. As a result, fluorescent AgNCs are being formed. Fluorescence is measured at excitation/emission wavelengths of 625/705 nm. The calibration plot for SA is linear in the 6 to 600 nM concentration range, with a 2.6 nM detection limit. The assay is simple, sensitive and affordable. Conceivably, the method may also be used to detect the binding of other small molecules to proteins.

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Graphical abstract A fluorescent sensing platform for small molecule-protein interaction assay has been developed based on terminal protection strategy and ssDNA-templated silver nanoclusters (AgNCs).

     

DNA-templated silver nanoclusters for fluorometric determination of the activity and inhibition of alkaline phosphatase

Microchimica Acta - The authors describe a fluorometric assay for determination of the activity and inhibition of alkaline phosphatase (ALP). It is based on the use of silver nanoclusters (AgNCs)...     

Fluorometric determination of DNA using nano-SiO2 particles as an effective dispersant and stabilizer for acridine orange

A sensitive fluorometric method for the determination of ctDNA (calf thymus DNA) is presented. It has good selectivity and sensitivity and uses nano-SiO₂ particles as an effective dispersant and stabilizer for acridine orange (AO). Compared to resonance light scattering (RLS) and the conventional method that uses organic dyes as fluorescence probe, the new method is more tolerant towards coexisting foreign substances and also more stable. With 20 mg nano-SiO₂ particles, 10 μmol L⁻¹ AO, at pH 8.01 and an ionic strength of 0.02 mol L⁻¹, the interaction of AO with nano-SiO₂ and ctDNA results in fluorescent signal enhancement. The extent of enhancement was in good proportion to the concentration of ctDNA at excitation/emission wavelengths of 490/523 nm, respectively. The calibration curve was linear over 0.66–55.60 μg mL⁻¹. The determination limit (3σ) was 15 μg mL⁻¹. The method was applied to the determination of ctDNA in synthetic samples with satisfactory results.     

Improvement of antibacterial activity of copper nanoclusters for selective inhibition on the growth of gram-positive bacteria

In general, copper nanoclusters (CuNCs) possess very low or even virtually no bactericidal effect. Herein,we report a novel CuNCs possessing significantly high antibacterial activity, that is tannic acid (TA)capped CuNCs (TA-CuNCs). TA-CuNCs exhibit strong absorption and excitation-dependent fluorescence within pH 2-12, resulting from the functional groups of TA-CuNCs due to two prototropic equilibria,phenolphenolate and carboxyliccarboxylate. There exists synergistic effect of TA and copper nanoclusters which endows TA-CuNCs remarkable antibacterial capability as a microbicide, as characterized by the effective inhibition on the growth of gram-positive bacteria by damaging the cell membrane. By incubating 1 x 10~7 CFU/mL of gram-positive bacteria Staphylococcus aureus and Bacillus subtilis with 30 μg/mL of TA-CuNCs for 10 min, the bacteria are completely inhibited, while under same conditions the viabilities of gram-negative bacteria Escherichia coli 0157:H7 and Pseudomonas aeruginosa remain 85.0%, 72.0%, respectively. In addition, TA-CuNCs exhibit low cytotoxicity and favorable biocompatibility demonstrated by standard methyl thiazolyl tetrazolium (MTT) assay with HepG2 and 293 Tcells, giving rise to cell viability of 94.2% for HepG2 and 96.7% for 293 T by incubating 10~6 cell/mL with 200 μg/mL of TA-CuNCs for 24 h. These results make TA-CuNCs a potential alternative as bactericide for infection treatment caused by gram-positive bacteria.     

Platinum nanofilm formation by EC-ALE via redox replacement of UPD copper: studies using in-situ scanning tunneling microscopy

The growth of Pt nanofilms on well-defined Au(111) electrode surfaces, using electrochemical atomic layer epitaxy (EC-ALE), is described here. EC-ALE is a deposition method based on surface-limited reactions. This report describes the first use of surface-limited redox replacement reactions (SLR(3)) in an EC-ALE cycle to form atomically ordered metal nanofilms. The SLR(3) consisted of the underpotential deposition (UPD) of a copper atomic layer, subsequently replaced by Pt at open circuit, in a Pt cation solution. This SLR(3) was then used a cycle, repeated to grow thicker Pt films. Deposits were studied using a combination of electrochemistry (EC), in-situ scanning tunneling microscopy (STM) using an electrochemical flow cell, and ultrahigh vacuum (UHV) surface studies combined with electrochemistry (UHV-EC). A single redox replacement of upd Cu from a PtCl(4)(2-) solution yielded an incomplete monolayer, though no preferential deposition was observed at step edges. Use of an iodine adlayer, as a surfactant, facilitated the growth of uniformed films. In-situ STM images revealed ordered Au(111)-(square root 3 x square root 3)R30 degrees-iodine structure, with areas partially distorted by Pt nanoislands. After the second application, an ordered Moiré pattern was observed with a spacing consistent with the lattice mismatch between a Pt monolayer and the Au(111) substrate. After application of three or more cycles, a new adlattice, a (3 x 3)-iodine structure, was observed, previously observed for I atoms adsorbed on Pt(111). In addition, five atom adsorbed Pt-I complexes randomly decorated the surface and showed some mobility. These pinwheels, planar PtI(4) complexes, and the ordered (3 x 3)-iodine layer all appeared stable during rinsing with blank solution, free of I(-) and the Pt complex (PtCl(4)(2-)).     

Fluorometric determination of cadmium(II) and mercury(II) using nanoclusters consisting of a gold-nickel alloy

A one-pot route has been developed for the preparation of bovine serum albumin-templated nickel-doped bimetallic gold-nickel nanoclusters (BSA-Au-Ni NCs) at a 10:1 M ratio of the precursor salts in a BSA matrix under alkaline conditions. The metal ions are reduced to the metal alloys by BSA. The resulting NCs display strong fluorescence and dual emission with peaks at 405 and 640 nm, respectively, under excitation at 340 nm. Fluorescence is strongly enhanced on addition of Cd(II) ions, but quenched on addition of Hg(II) ions. The findings have been exploited to design a fluorometric method for the separate determination of Cd(II) and Hg(II), respectively. The optimized analytical nanosystem displays relatively good dynamics between enhancement and quenching. Cd(II) and Hg(II) can be quantified in the 0 to 200 and 0 nM to 24 μM, respectively. The limits of detection are ~1.8 nM in both cases, which indicates the highest sensitivity to Cd(II) and Hg(II) ions for a fluorescent probe. This new kind of nanocrystal probe is hardly interfered by a range of commonly encountered metal ions. Its advantages were demonstrated by determining Cd(II) and Hg(II) ions in spiked serum samples.

Dually emitting nanoclusters composed of gold-nickel alloys are shown to act as very sensitive fluorescent probes for the detection of Cd(II) and Hg(II) ions.

     

A highly selective colorimetric sulfide assay based on the inhibition of the peroxidase-like activity of copper nanoclusters

The authors report that sulfide ions are capable of inhibiting the peroxidase-like activity of copper nanoclusters (CuNCs). The catalytic activity of CuNCs toward the oxidation of the chromogenic substrate 3,3′,5,5′-tetramethylbenzidine by H₂O₂ is remarkably decreased in the presence of sulfide. Based on this finding, a colorimetric assay was developed for the rapid determination of sulfide. Best operated at a wavelength of 652 nm, it has a 0.5 μM detection limit. The method is highly selective and has been successfully applied to the quantification of sulfide in environmental water samples.

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Graphical abstract The catalytic activity of CuNCs toward the oxidation of 3,3′,5,5′-tetramethylbenzidine by H₂O₂ is remarkably decreased in the presence of sulfide ions. This finding has been applied to design a method for colorimetric quantification of sulfide ions in environmental samples.

     

Use of immobilized triazine dyes in the purification of DNA topoisomerase I (Topo I) and terminal deoxynucleotidyl transferase (TdT) from calf thymus.

The interaction between TdT and Topo I, and twelve various triazine dyes immobilized on Sepharose CL-6B was studied. Yellow lightproof 2KT-Sepharose and Bordeaux 4ST-Sepharose were used to purify TdT and Topo I, respectively. The principal role of copper ions, complexed to the dye molecules, in the dye-protein interaction was evaluated.     

Iron/copper promoted oxidative homo-coupling reaction of terminal alkynes using air as the oxidant

An inexpensive catalytic system, which used a readily available Fe(acac)₃ and trace quantity of Cu(acac)₂ as the co-catalyst and air as the oxidant for the homo-coupling of terminal alkynes, has been developed. The catalytic system could also apply to the cross-coupling reaction of two different terminal alkynes.     

Photometric determination of the activity of cellulase and xylanase via measurement of formation of gold nanoparticles

We describe a colorimetric assay for the determination of the activity of cellulase and xylanase. Following enzymatic hydrolysis, reductive saccharides are produced which are capable of directly reducing auric acid to form gold nanoparticles (AuNPs). The AuNPs are of fuchsia color and possess a strong plasmonic absorption band at 550 nm. Reaction conditions such as temperature, reaction time, and pH of the solution were optimized. A linear relationship between the concentrations of saccharide and the plasmon absorption of gold nanoparticles at 550 nm allowed for quantitative detection of the saccharides formed in solution, from which the hydrolase activity can be calculated. The detection limits for cellulase and xylanase are 0.14 and 0.080 IU mL^?1. The results were compared with those of the 3,5-dinitrosalicylic acid method and showed the established method to be reliable and accurate.

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Graphical abstract Following enzymatic hydrolysis, reductive saccharides are produced which are capable of directly reducing auric acid to form colloidal gold. The plasmon absorption of the colloidal gold is directly proportional to the amount of reductive saccharides, which can be used to calculate the activity of hydrolase indirectly.

     

Fluorometric determination of DNA using a new ruthenium complex Ru(bpy)2PIP(V) as a nucleic acid probe.

A new ligand, 2-phenyl(5-fluoro)imidazo[f]-1,10-phenanthroline (PIP(V)), and its coordination compounds, Ru(bpy)2PIP(V), were synthesized. The fluorescence spectrum of the interaction between Ru(bpy)2PIP(V) and DNA was studied, and a very strong fluorescence peak at a wavelength of 589 nm appeared. The optimum condition of analyzing DNA was decided. The method is simple, convenient and fast, and also has high sensitivity and good selectivity. It has been satisfactorily employed for determinations in synthesized samples.     

Synthesis of cysteine-functionalized water-soluble luminescent copper nanoclusters and their application to the determination of chromium(VI)

A facile, one-pot green method is presented for the preparation of water-soluble luminescent copper nanoclusters (Cu-NCs) from copper dichloride and cysteine as the precursor and stabilizer, respectively. The Cu-NCs are characterized by high resolution transmission electron microscopy, X-ray photoelectron spectroscopy, fluorescence, UV–Vis, and Raman spectroscopy. The Cu-NCs have an average size of 3.5 nm and are stable in aqueous solution at least for 2 weeks. Under photo excitation with 365 nm light, the Cu-NCs display strong green fluorescence with the maximum of emission at 490 nm and a quantum yield of 5.6 %. Fluorescence is quenched by Cr(VI) ion, and this effect was exploited to develop a highly selective method for the determination of Cr(VI). The detection limit of this probe is as low as 43 nM.

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Graphical Abstract A facile, one-pot, “green” synthetic route was developed for preparing water-soluble luminescent copper nanoclusters (CuNCs) by using copper chloride and cysteine as the precursor and stabilizer, respectively. Their fluorescence is quenched by Cr(VI) ion, and this is exploited in a sensitive assay for Cr(VI) ions.

     

A rapid and simple HPLC-UV method for the determination of inhibition characteristics of farnesyl transferase inhibitors

Ras proteins play an important role in the development of cancer. Farnesyl transferase inhibitors (FTIs) block the first obligatory post-translational step for activation, prenylation, of Ras proteins. To find new potent FTIs, rapid enzyme activity assays are required to reduce FTI development time. Most assays to date are based on radioactive labelled substrates. We developed a new, in vitro, farnesyl transferase assay based on gradient chromatography coupled to UV detection. Unfarnesylated and farnesylated H-Ras proteins were resolved on a C18 wide-pore HPLC column and their concentrations were determined with use of a calibration curve of unfarnesylated H-Ras. The assay was used to investigate inhibition characteristics of FTIs. The IC50 values of the FTIs L778,123 and SCH66336 were 4.2 nm and 78 microm, respectively. This assay could support the screening and development of FTIs to obtain rapid insights into their inhibitory properties.