De-Ming Kong

Ag+ and Cysteine Quantitation Based on G-Quadruplex−Hemin DNAzymes Disruption by Ag+

Some G-quadruplex-hemin complexes are DNAzyme peroxidases that efficiently catalyze H(2)O(2)-mediated reactions, such as the oxidation of ABTS (2,2-azinobis(3-ethylbenzothiozoline)-6-sulfonic acid) by H(2)O(2). Since Ag(+) chelates guanine bases at the binding sites are involved in G-quadruplex formation, the presence of Ag(+) may disrupt these structures and inhibit the peroxidase activity of G-quadruplex-hemin DNAzymes. On the basis of this principle, a highly sensitive and selective Ag(+)-detection method was developed. The method allows simple detection of aqueous Ag(+) with a detection limit of 64 nM and a linear range of 50-3000 nM. Cysteine (Cys) is a strong Ag(+)-binder and competes with quadruplex-forming G-rich oligonucleotides for Ag(+)-binding, promoting the reformation of G-quadruplexes and increasing their peroxidase activity. Therefore, the Ag(+)-sensing system was also developed as a Cys-sensing system. This turn-on process allowed the detection of Cys at concentrations as low as 50 nM using a simple colorimetric technique. The Cys-sensing system could also be used for the detection of reduced glutathione (GSH). Neither the Ag(+)-sensing nor the Cys-sensing systems required labeled oligonucleotides. In addition, both gave large changes in absorbance signal that could be observed by the naked eye. Thus, a simple visual method for Ag(+)- or Cys-detection was developed.

Two luminescent metal–organic frameworks for the sensing of nitroaromatic explosives and DNA strands

With a flexible aromatic ligand, bis-(3,5-dicarboxy-phenyl)terephthalamide (H4L), two metal–organic frameworks, Cd(L)·(HDMA)2(DMF)(H2O)3 (1) and Zn(L)·(HDMA)2(DMF)(H2O)6 (2), were synthesized using a solvothermal method. Both 1 and 2 possess a 3D open framework. These two compounds exhibit strong photoluminescence in suspensions, and their luminescence could be efficiently and selectively quenched by a series of nitroaromatics. Thus, 1 and 2 could be used as excellent luminescent probes for nitroaromatic explosives. Additionally, 1 and 2 could also be utilized as the sensing platforms for DNA strands, which is attributed to their different affinities for single- and double-stranded DNAs. With this method, 0.05 nM of target DNA could be detected, and the selectivity could be down to a single nucleotide mismatch. This work shows the potential of MOFs for the sensing of versatile chemicals.

Oxidative DNA cleavage by Schiff base tetraazamacrocyclic oxamido nickel(II) complexes

Nickel is considered a weak carcinogen. Some researches have shown that bound proteins or synthetic ligands may increase the toxic effect of nickel ions. A systematic study of ligand effects on the interaction between nickel complexes and DNA is necessary. Here, we compared the interactions between DNA and six closely related Schiff base tetraazamacrocyclic oxamido nickel(II) complexes NiL(1-3a,1-3b). The structure of one of the six complexes, NiL(3b) has been characterized by single crystal X-ray analysis. All of the complexes can cleave plasmid DNA under physiological conditions in the presence of H(2)O(2). NiL(3b) shows the highest DNA cleavage activity. It can convert supercoiled DNA to nicked DNA then linear DNA in a sequential manner as the complex concentration or reaction time is increased. The cleavage reaction is a typical pseudo-first-order consecutive reaction with the rate constants of 3.27+/-0.14h(-1) (k(1)) and 0.0966+/-0.0042h(-1) (k(2)), respectively, when a complex concentration of 0.6mM is used. The cleavage mechanism between the complex and plasmid DNA is likely to involve hydroxyl radicals as reactive oxygen species. Circular dichronism (CD), fluorescence spectroscopy and gel electrophoresis indicate that the complexes bind to DNA by partial intercalative and groove binding modes, but these binding interactions are not the dominant factor in determining the DNA cleavage abilities of the complexes.

G-quadruplex–hemin DNAzyme-amplified colorimetric detection of Ag+ ion

A G-quadruplex-hemin DNAzyme-amplified Ag(+)-sensing method was developed based on the ability of Ag(+) to stabilize C-C mismatches by forming C-Ag(+)-C base pairs. In this method, only one unlabelled oligonucleotide strand was used. In the absence of Ag(+), the oligonucleotide strand formed an intramolecular duplex. The G-rich sequence in the oligonucleotide was partially caged in this duplex structure and cannot fold into the G-quadruplex structure. The addition of Ag(+) promoted the formation of another intramolecular duplex in which C-C mismatches were stabilized by C-Ag(+)-C base pairs, leading to the release of the G-rich sequence which can fold into a G-quadruplex capable to bind hemin to form a catalytically active G-quadruplex-hemin DNAzyme. As a result, a UV-vis absorbance increasing was observed in the H(2)O(2)-ABTS (2,2-azinobis(3-ethylbenzothiozoline)-6-sulfonic acid) reaction system. This turn-on process allowed the detection of aqueous Ag(+) at concentrations as low as 6.3 nM using a simple colorimetric technique, showing a high selectivity over a range of other metal ions.

A New Cationic Porphyrin Derivative (TMPipEOPP) with Large Side Arm Substituents: A Highly Selective G-Quadruplex Optical Probe

The discovery of uncommon DNA structures and speculation about their potential functions in genes has brought attention to specific DNA structure recognition. G-quadruplexes are four-stranded nucleic acid structures formed by G-rich DNA (or RNA) sequences. G-rich sequences with a high potential to form G-quadruplexes have been found in many important genomic regions. Porphyrin derivatives with cationic side arm substituents are important G-quadruplex-binding ligands. For example, 5,10,15,20-Tetrakis(N-methylpyridinium-4-yl)-21H,23H-porphyrin (TMPyP4), interacts strongly with G-quadruplexes, but has poor selectivity for G-quadruplex versus duplex DNA. To increase the G-quadruplex recognition specificity, a new cationic porphyrin derivative, 5,10,15,20-tetra-{4-[2-(1-methyl-1- piperidinyl)ethoxy]phenyl} porphyrin (TMPipEOPP), with large side arm substituents was synthesized, and the interactions between TMPipEOPP and different DNA structures were compared. The results show that G-quadruplexes cause large changes in the UV-Vis absorption and fluorescence spectra of TMPipEOPP, but duplex and single-stranded DNAs do not, indicating that TMPipEOPP can be developed as a highly specific optical probe for discriminating G-quadruplex from duplex and single-stranded DNA. Visual discrimination is also possible. Job plot and Scatchard analysis suggest that a complicated binding interaction occurs between TMPipEOPP and G-quadruplexes. At a low [G-quadruplex]/[TMPipEOPP] ratio, one G-quadruplex binds two TMPipEOPP molecules by end-stacking and outside binding modes. At a high [G-quadruplex]/[TMPipEOPP] ratio, two G-quadruplexes bind to one TMPipEOPP molecule in a sandwich-like end-stacking mode.

Photoluminescent sensing for acidic amino acids based on the disruption of graphene quantum dots/europium ions aggregates

A simple mix-and-detect photoluminescence method was developed for the turn-on detection of acidic amino acids. To achieve this, graphene quantum dots (GQDs), which emit both down-conversion and up-conversion photoluminescence were prepared by solvothermal synthesis. The carboxylic acid-rich surface not only increases the water solubility of the prepared GQDs, but also makes Eu(3+)-triggered GQDs aggregation possible, thus causing the photoluminescence quenching of GQDs. The quenched photoluminescence can be recovered by the competition between acidic amino acids and GQDs for Eu(3+). Under optimized conditions, sensitive and specific acidic amino acids quantitation can be achieved by utilizing the changes in either down-conversion or up-conversion photoluminescence. Up-conversion mode gives a little lower detection limit than the down-conversion one. Nearly overlapped calibration curves were obtained for the two acidic amino acids, glutamic acid (Glu) and aspartic acid (Asp), thus suggesting that the proposed method can be used not only for the quantitation of individual acidic amino acids, but also for the detection of total amount of them.

Duplex probes: a new approach for the detection of specific nucleic acids in homogenous assays

A novel method for the detection of specific nucleic acids in homogenous solution was developed. The method is based on the use of duplex probes in which fluorescent donor and quencher labeled on either oligonucleotide are held in close proximity, so that fluorescence is quenched. Amplification of the target sequence results in the cleavage of the probe and the resulting fluorescence can be detected. The fluorescent data analysis demonstrated that the duplex probes can specifically recognize the presence of target, and a significantly higher lever of relative fluorescent signal than TaqMan probes is obtainable. Combined with real-time PCR instruments, the assay can be used to quantify the input target molecules. As few as five copies of initial target molecules can be detected, and a large dynamic linear ranger (five orders of magnitude) is obtained.

Sequence-specific detection of nucleic acids utilizing isothermal enrichment of G-quadruplex DNAzymes

G-quadruplex DNAzymes are peroxidase-like complexes formed by nucleic acid G-quadruplexes and hemin. Various chemical sensors and biosensors have been developed, based on such DNAzymes. Here we report a novel, specific nucleic acid detection method utilizing the isothermal amplification strategy of G-quadruplex DNAzymes. In this method, an unlabeled oligonucleotide probe was used. The probing sequence of the oligonucleotide was in the form of a stem-loop structure. A G-rich sequence, containing three GGG repeats, was linked to the 5-end of the stem-loop structure. In the presence of target, the probing sequence hybridized to the target, and a G(n) (n≥2) repeat was extended from its 3-end. This G(n) repeat, together with the three GGG repeats at the 5-end, folded into a G-quadruplex, and displayed enhanced peroxidase acitivity upon hemin binding. Utilizing the dynamic binding interaction between the probe and its target, the enrichment of G-quadruplex DNAzymes was achieved. Using this method, simple, rapid and cost-effective nucleic acid detection could be achieved. This method displayed high target-length tolerance and good detection specificity; one-base mismatch could be judged easily, even by visual inspection. This method may be used as an auxiliary tool for amplified detection of specific DNA targets in some situations, in which isothermal detection is desirable.

PCR hot-start using duplex primers

A new technique of PCR hot-start using duplex primers has been developed which can decrease the undesirable products arising throughout PCR amplification thereby giving better results than a manual hot-start method.

Applications of porous organic frameworks (POFs) in detection of nucleic acid and exonuclease I activity

As a two-dimensional (2D) ordered porous organic framework (POF), PAF-6 is demonstrated to have an extraordinarily high fluorescence quenching ability to dye-labeled single-stranded DNA (ssDNA). Based on its different affinities to ssDNA and double-stranded DNA (dsDNA), and to ssDNAs with different lengths, PAF-6 is firstly utilized as a simple, cost-efficient, sensitive and selective sensing platform for sequence-specific detection of DNA and activity analysis of exonuclease I (Exo I). In these two systems, the sensing approach is accomplished by simply mixing the dye-labeled ssDNA probe with the targets and PAF-6. The targets of DNA and Exo I are specifically and sensitively detected with detection limits of 0.6 nM and 0.03 U mL-1 (S/N = 3), respectively, by using PAF-6 as a fluorescence quencher of the dye-labeled ssDNA probe. The results of this study suggest that PAF-6 can be developed as an excellent platform for the detection of nucleic acid and nuclease activity. In addition, PAF-6 exhibits a remarkable ability to protect ssDNA probe from enzymatic digestion, which may greatly extend the applications of the proposed ssDNA probe/PAF-6 sensing system to bioanalysis and biomedicine.