Xiang Zhou

Highly Effective Colorimetric and Visual Detection of Nucleic Acids Using an Asymmetrically Split Peroxidase DNAzyme

G-quadruplex containing peroxidase DNAzyme is a complex of hemin and a single-stranded guanine-rich DNA (hemin-binding DNA aptamer), which is used as an attractive catalytic label for biosensing recently. Therein, the hemin-binding DNA aptamer contains four GGG repeats and can fold into a G-quadruplex structure. In this paper, we have developed a new split mode to divide the hemin-binding DNA aptamer into two parts: one possesses three GGG repeats, and another part possesses one GGG repeat, namely, the 3:1 split mode. The combination of G-quadruplex and hemin binding could be used as a sensitive probe for the identification of single nucleotide polymorphisms by giving a color signal, visible to the naked eye at room temperature. The G-quadruplex containing peroxidase DNAzyme utilizes the 3:1 split mode and can be directly used for the identification of SNPs with a detection limit in the nM range when the matching length of the probe is short enough. When the matching length of the probe is relatively long, another method adding competition sequences to the probe could also operate effectively for the identification of SNPs. The results also suggested that we could detect the signal when the mutation sample was only 5% in the total target DNA with a competition strategy.

Sensitive and Convenient Detection of microRNAs Based on Cascade Amplification by Catalytic DNAzymes

On target: We have developed two cascade amplification strategies that combine duplex specific nuclease (DSN) amplicon with either G-quadruplex-based DNA peroxidase or 8-17 DNAzyme amplicon for miRNA detection. In this way, sensitive and convenient detection of miRNAs was achieved. In the DNA peroxidase-based system, a visual color change could be observed in the presence of target miRNAs (see scheme).

Existence of G-quadruplex structures in promoter region of oncogenes confirmed by G-quadruplex DNA cross-linking strategy

Existence of G-quadruplex DNA in vivo always attract widespread interest in the field of biology and biological chemistry. We reported our findings for the existence of G-quadruplex structures in promoter region of oncogenes confirmed by G-quadruplex DNA cross-linking strategy. Probes for selective G-quadruplex cross-linking was designed and synthesized that show high selectivity for G-quadruplex cross-linking. Further biological studies demonstrated its good inhibition activity against murine melanoma cells. To further investigate if G-quadruplex DNA was formed in vivo and as the target, a derivative was synthesized and pull-down process toward chromosome DNAs combined with circular dichroism and high throughput deep sequencing were performed. Several simulated intracellular conditions, including X. laevis oocytes, Ficoll 70 and PEG, was used to investigate the compounds pure cross-linking ability upon preformed G-quadruplex. Thus, as a potent G-quadruplex cross-linking agent, our strategy provided both valuable evidence of G-quadruplex structures in vivo and intense potential in anti-cancer therapy.

DNA methyltransferase activity detection based on fluorescent silver nanocluster hairpin-shaped DNA probe with 5’-C-rich/G-rich-3’ tails

DNA methylation has received a large amount of attention due to its close relationship to a wide range of biological phenomena, such as gene activation, gene imprinting, and chromatin stability. Herein, we have designed a hairpin-shaped DNA probe with 5-C-rich/G-rich-3 tails and developed a simple and reliable fluorescence turn-off assay for DNA adenine methylation (Dam) methyltransferase (MTase) detection combining site recognition and the fluorescence enhancement of DNA-templated silver nanoclusters (DNA-AgNCs) by guanine-rich DNA sequences. A designed hairpin probe with 5 CCCTTACCCC and 3 GGGTGGGGTGGGGTGGGG displays a bright red emission after reacting with AgNO3 and NaBH4. In the presence of Dam MTase, the methylation-sensitive restriction endonuclease Dpn I which has the same recognition site with the Dam MTase can split the probe, freeing the G-rich sequence from the C-rich sequence, thus quenching the fluorescence of DNA-AgNCs. Compared to traditional fluorescent-based methods, this strategy is simple and inexpensive. A linear response to concentrations of Dam MTase which range from 1 U/mL to 100 U/mL and a detection limit of 1 U/mL are obtained without any amplification steps. In addition, we also demonstrate the method can be used for evaluation and screening of inhibitors for Dam MTase.

The Sensitive and Selective Optical Detection of Mercury(II) Ions by Using a Phosphorothioate DNAzyme Strategy

Guanine-rich DNA sequences are widely found in telomere regions and are prone to forming G-quadruplex structures through cyclic Hoogsteen base pairing between four guanine bases. According to the orientation of oligonucleotides, three formations of G-quadruplex have been suggested: all-parallel, antiparallel, and a hybrid of parallel/antiparallel, all of which have been reported as existing under different conditions. Conformations of the DNA quadruplex were deemed to be affected by several factors such as the composition of the loops, the number of stacked G-tetrads, and the presence of diverse cations or small molecules. Directly modified oligonucleotides, which could be utilized to form a G-quadruplex, have been found, for example, by Hartig and co-workers. The main aim of the study reported herein was to develop a new approach to form G-quadruplex complexes more easily and conveniently by modification of the sequence and to subsequently make use of these conformations as functional probes. Phosphorothioate oligonucleotides are a variant of normal DNA and are characterized by the replacement of a nonbridging oxygen group with a sulfur. This modification facilitates oligonucleotide stability in the presence of endoand exo-nucleases, which could be used in gene regulation through the antisense approach. In addition, recent investigations by Deng and co-workers have indicated that the phosphorothioation of DNA exist in some bacteria, which could be considered as the first known physiological modification on the DNA backbone. To develop this strategy, we combined the two principles that oligonucleotides could be modified with sulfur, and that the formation of phosphorothioate G-quadruplex could be regulated by metal ions, such as Hg , since Hg S forms a strongly covalent bond that would result in the folding of the G-quadruplex. Furthermore, the modified G-quadruplex could be utilized as a functional tool to identify potential targets of anticancer therapy, as well as the detection of specific metal ions or the identification of single-nucleotide polymorphisms (SNPs) as a biological probe. The toxicity of Hg to humans and the environment is associated with its disruption of cell membranes, its impairment of mitochondrial function, and its inhibition of DNA replication in a cell. Up to now, several methods have been developed for the detection of Hg under aqueous conditions with high sensitivity and selectivity, such as the application of DNAzymes and oligonucleotide–gold nanoparticles. The peroxidase-like DNAzyme comprising a G-quadruplex structure and hemin was first designed and developed by Sen and co-workers. Then Willner and co-workers applied it to detect small molecules, metal ions, proteins, and DNA as a catalytic label. Thereafter, Mikuma et al. reported that the tetramolecular G-quadruplex formed by dACHTUNGTRENNUNG(TTAGGG) possessed the ability to bind with hemin. In our strategy, the phosphorothioate modification was adopted within one of the T4G4 sequences, which were inclined to form G-quadruplex structures, and then utilized it to form a DNAzyme (Scheme 1). Compared to the former detection methods of Hg , the utilization of the DNAzyme operated by the phosphorothioate all-parallel G-quadruplex formed from T4G4-S3 combined the advantage of sensitive and selective optical detec[a] D. Zhang, M. Deng, L. Xu, Y. Zhou, J. Yuwen, Prof. X. Zhou College of Chemistry and Molecular Sciences Wuhan University, Hubei Wuhan 430072 (China) Fax: (+86) -27-87336380 E-mail : xzhou@whu.edu.cn [b] Prof. X. Zhou Key Laboratory of Biomedical Polymers of Ministry of Education College of Chemistry and Molecular Sciences Wuhan University, Hubei Wuhan, 430072 (China) [c] Prof. X. Zhou The State Key Laboratory of Natural and Biomimetic Drugs Beijing University (China) [] The two authors contributed equally to this work. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.200901268.

Selective chemical labelling of 5-formylcytosine in DNA by fluorescent dyes.

Direct labelling: 5-Formylcytosine in DNA can be selectively labelled by fluorescent dyes containing an active amino group. The labelled DNA shows strong fluorescence and can be detected by polyacrylamide gel electrophoresis (PAGE) and fluorescence measurements (see scheme). This method can distinguish 5-formylcytosine from other methylation forms of cytosine in DNA.

Selective detection of 5-formyl-2'-deoxycytidine in DNA using a fluorogenic hydroxylamine reagent.

Fluorogenic hydroxylamine reagents were used for detecting 5-fC through a labeling pathway. Chemical synthesis, HPLC, denaturing PAGE, and DNA MS were applied to testify that the probe reacted with 5-fC with oligodeoxynucleotide selectivity to achieve 5-fC detection conveniently and quantificationally with the method of fluorescence. The feasibility of fluorescently detecting 5-fC in a genome was also investigated.

Hydrophilic Material for the Selective Enrichment of 5-Hydroxymethylcytosine and Its Liquid Chromatography–Tandem Mass Spectrometry Detection

5-Methylcytosine (5-mC), an important epigenetic modification involved in development, can be converted enzymatically to 5-hydroxymethylcytosine (5-hmC). 5-hmC is considered an intermediate of active DNA cytosine demethylation and makes itself serve as an epigenetic mark. 5-hmC content in most mammalian cells is low and the quantification of 5-hmC by liquid chromatography-mass spectrometry (LC-MS) frequently suffers from ion suppression by the presence of unmodified nucleosides. To circumvent this problem, we developed a method to selectively transfer a glucosyl group to the hydroxymethyl moiety of 5-hmC and form a more hydrophilic residue (β-glucosyl-5-hydroxymethyl-2-deoxycytidine, 5-gmdC) by using T4 β-glucosyltransferase. The more hydrophilic 5-gmdC can be selectively enriched by using NH2-silica via hydrophilic interaction prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, which eliminates the ion suppression and significantly improves the detection sensitivity and accuracy. Using this method, we successfully quantified 5-hmC content in genomic DNA of three human cell lines and seven yeast strains. To the best of our knowledge, this is the first report about the existence of 5-hmC in the model organism of yeast. In addition, the contents of 5-hmC in two yeast strains of Schizosaccharomyces pombe are even higher than those of 5-mC, indicating that 5-hmC may play important roles on the physiological functions of yeast.

Highly sensitive detection of telomerase based on a DNAzyme strategy.

The present study demonstrated a highly sensitive strategy for measuring telomerase activity in cell extracts. Furthermore, we applied the new strategy for in situ detection of telomerase at the cellular level in cancer cells, together with a normal cell as the negative control.

A two-photon fluorescent probe for intracellular detection of tyrosinase activity.

AAN effective sensor: The two-photon turn-on fluorescent probe NHU was synthesized to optically detect tyrosinase activity in vitro and in melanoma cells. NHU is composed of a 4-aminophenol moiety and a naphthylamine unit, both of which are connected through a urea linkage. Upon exposure to tyrosinase, the 4-aminophenol site is gradually oxidized to the corresponding orthoquinone, ultimately releasing the highly fluorescent product 6-acyl-N-methyl-2-naphthylamine (AAN).