Shaoru Wang

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.

Novel Amplex Red Oxidases Based on Noncanonical DNA Structures: Property Studies and Applications in MicroRNA Detection

G-triplex has recently been identified as a new secondary structure in G-rich sequences. However, its functions and biological roles remain largely unknown. This study first developed two kinds of Amplex Red oxidases, which were based on relatively new G-triplex structure and a common G-quadruplex one. A collection of DNA binding assays including circular dichroism (CD) spectroscopy, a CD melting assay, and a UV titration study were used to determine the G-triplex structure of G3 oligomer. The low intrinsic oxidative activity of hemin was significantly enhanced using G-triplex or G-quadruplex. Only one key guanine deletion from the G3 oligomer or G4 one could result in a much decreased Amplex Red oxidation activity. To the best of our knowledge, this is the first case reporting direct use of air as the oxidant for fluorescence generation based on DNAzyme strategies. Further mechanism studies demonstrated an involvement of on-site H2O2 generation from O2 and water and a following oxidation of Amplex Red to resorufin, causing a fluorescence enhancement. Furthermore, the newly developed oxidases have been effectively used in microRNA detection, using only one biotin-labeled probe and one small-molecule substrate. The conjugation of a target DNA to the G-triplex- or G-quadruplex-forming sequence enabled one to produce G-triplex or G-quadruplex by endonuclease in the presence of a slight amount of miRNA and amplify the signal of fluorescence from the oxidation of Amplex Red. Our findings of novel Amplex Red oxidases could potentially be used in a wide range of applications.

A highly conserved G-rich consensus sequence in hepatitis C virus core gene represents a new anti-hepatitis C target.

A conserved guanine-rich sequence could be a new target for anti–hepatitis C virus drug development. G-quadruplex (G4) is one of the most important secondary structures in nucleic acids. Until recently, G4 RNAs have not been reported in any ribovirus, such as the hepatitis C virus. Our bioinformatics analysis reveals highly conserved guanine-rich consensus sequences within the core gene of hepatitis C despite the high genetic variability of this ribovirus; we further show using various methods that such consensus sequences can fold into unimolecular G4 RNA structures, both in vitro and under physiological conditions. Furthermore, we provide direct evidences that small molecules specifically targeting G4 can stabilize this structure to reduce RNA replication and inhibit protein translation of intracellular hepatitis C. Ultimately, the stabilization of G4 RNA in the genome of hepatitis C represents a promising new strategy for anti–hepatitis C drug development.

Chemical Targeting of a G-Quadruplex RNA in the Ebola Virus L Gene

In the present study, our bioinformatics analysis first reveals the existence of a conserved guanine-rich sequence within the Zaire ebolavirus L gene. Using various methods, we show that this sequence tends to fold into G-quadruplex RNA. TMPyP4 treatment evidently inhibits L gene expression at the RNA level. Moreover, the mini-replicon assay demonstrates that TMPyP4 effectively inhibits the artificial Zaire ebolavirus mini-genome and is a more potent inhibitor than ribavirin. Although TMPyP4 treatment reduced the replication of the mutant mini-genome when G-quadruplex formation was abolished in the L gene, its inhibitory effect was significantly alleviated compared with wild-type. Our findings thus provide the first evidence that G-quadruplex RNA is present in a negative-sense RNA virus. Finally, G-quadruplex RNA stabilization may represent a new therapeutic strategy against Ebola virus disease.

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.

Visual Detection of rpoB Mutations in Rifampin-Resistant Mycobacterium tuberculosis Strains by Use of an Asymmetrically Split Peroxidase DNAzyme

ABSTRACT Multidrug-resistant Mycobacterium tuberculosis is resistant to two first-line antituberculosis drugs, isoniazid and rifampin, resulting in the relapse of tuberculosis. M. tuberculosis grows very slowly, and thus traditional examination methods take time to test its drug resistance and cannot meet clinical needs. The use of a DNA probe makes it possible to test rifampin resistance. We developed an asymmetrical split-assembly DNA peroxidase assay to detect drug-resistant mutation of rifampin-resistant M. tuberculosis in the rpoB gene rapidly and visibly. A new strategy was also designed to eliminate the adverse effects caused by the complicated secondary structure of the target DNA and to improve the efficiency of the probes. This detection system consists of five group detections, covers rifampin-resistant determination region of the rpoB gene, and tests 40 kinds of mutations, including the most common mutations at codons 531 and 526. Every group detection or individual mutant allele detection can distinguish corresponding mutant DNA sequences from the wild-type DNA sequences.

Cationic Porphyrins and Analogues as New DNA Topoisomerase I and II Inhibitors

A series of cationic porphyrins, and analogues such as cationic corroles and phthalocyanines were found to have biological activities towards topoisomerases I and II in vitro. Cationic porphyrins and phthalocyanines do not induce Topo I-DNA covalent complexes but inhibit topoisomerase I by direct binding to DNA, which limits topoisomerase I access to the DNA substrate. The lowest concentration where an inhibition effect is clearly visible of some derivatives is between 0.1 and 0.6 microM. Furthermore, some complexes were found to inhibit the activity of the topoisomerase II.

Systematic investigations of different cytosine modifications on CpG dinucleotide sequences: the effects on the B-Z transition.

We have first demonstrated the distinctive effects of three newly reported epigenetic modifications, including 5hmC, 5fC, and 5caC, on B-Z transition of CpG dinucleotide DNAs. We have performed detailed assays and compared their effects. We further studied the regulation of B-Z transition of CpG dinucleotide dodecamers by alternating oxidation and alternating reduction.

Small-Molecule-Triggered and Light-Controlled Reversible Regulation of Enzymatic Activity

The fine control of enzyme activity is essential for the regulation of many important cellular and organismal functions. The light-regulation of proteins serves as an important method for the spatiotemporal control over the production and degradation of an enzyme product. This area is of intense interest for researchers. To the best of our knowledge, the use of small molecules as light-triggered molecular switches to reversibly control enzyme activity at the protein level has not yet been studied. In the present study, we demonstrate the light-controlled reversible regulation of the enzyme using a small-molecule-triggered switch, which is based on molecular recognition between an azobenzene derivative and telomere DNA. This molecule interconverts between the trans and cis states under alternate 365 nm UV and visible light irradiation, which consequently triggers the compaction and extension of telomere DNA. We further provide direct evidence for this structural switch using a circular dichroism study. Furthermore, our strategy has been successfully used to effectively control blood clotting in human plasma.