Li-Xia Wang

Thioflavin T as an efficient fluorescence sensor for selective recognition of RNA G-quadruplexes

RNA G-quadruplexes (G4s) play important roles in translational regulation, mRNA processing events and gene expression. Therefore, a fluorescent probe that is capable of efficiently recognizing RNA G-quadruplex structures among other RNA forms is highly desirable. In this study, a water-soluble fluorogenic dye (i.e., Thioflavin T (ThT)) was employed to recognize RNA G-quadruplex structures using UV–Vis absorption spectra, fluorescence spectra and emission lifetime experiments. By stacking on the G-tetrad, the ThT probe exhibited highly specific recognition of RNA G-quadruplex structures with striking fluorescence enhancement compared with other RNA forms. The specific binding demonstrates that ThT is an efficient fluorescence sensor that can distinguish G4 and non-G4 RNA structures.

Directly lighting up RNA G-quadruplexes from test tubes to living human cells

RNA G-quadruplexes (G4s) are one of the key components of the transcriptome that act as efficient post-transcriptional regulatory elements in living cells. To conduct further studies of the unique biological functions of RNA G4s, techniques need to be developed that can efficiently recognize RNA G4 structures under various conditions, in fixed cells and living cells, as well as in vitro. This paper presents the development of such a method, a new technique using a cyanine dye called CyT, which can detect both canonical and non-canonical RNA G4 structures from test tubes to living human cells. The ability of CyT to distinguish between G4 and nonG4 RNA offers a promising tool for future RNA G4-based biomarker discovery and potential diagnostic applications.

Stabilization of G-quadruplex DNA and inhibition of Bcl-2 expression by a pyridostatin analog

The G-quadruplexes located in the P1 promoter of B-cell lymphoma-2 (Bcl-2) gene are implicated to regulate Bcl-2 expression. Here, we designed a new pyridostatin analog named PDF, which exhibited high specificity and stabilizing effect toward G-quadruplexes. The luciferase assay demonstrated that PDF could significantly suppress Bcl-2 transcriptional activation in human laryngeal squamous carcinoma cells (Hep-2) cells. Besides, PDF also induced cell apoptosis in vitro assays. These results provide an excellent G-quadruplex specific ligand as an efficient Bcl-2 inhibitor. These results also implicate that PDF may be a potential anticancer drug to head neck cancer.

Novel dual-functional regulation of a chair-like antiparallel G-quadruplex inducing assembly- disassembly of a cyanine dye†

Biomolecules are promising templates that play important roles in controlling the supramolecular assembly process more flexibly and precisely due to their unique characteristic structures. Here we first present a G-quadruplex with a chair-like antiparallel motif as a novel dual-functional regulation template in the aggregation of a cyanine dye.

Serum Metabolomics of Burkitt Lymphoma Mouse Models

Burkitt lymphoma (BL) is a rare and highly aggressive type of non-Hodgkin lymphoma. The mortality rate of BL patients is very high due to the rapid growth rate and frequent systemic spread of the disease. A better understanding of the pathogenesis, more sensitive diagnostic tools and effective treatment methods for BL are essential. Metabolomics, an important aspect of systems biology, allows the comprehensive analysis of global, dynamic and endogenous biological metabolites based on their nuclear magnetic resonance (NMR) and mass spectrometry (MS). It has already been used to investigate the pathogenesis and discover new biomarkers for disease diagnosis and prognosis. In this study, we analyzed differences of serum metabolites in BL mice and normal mice by NMR-based metabolomics. We found that metabolites associated with energy metabolism, amino acid metabolism, fatty acid metabolism and choline phospholipid metabolism were altered in BL mice. The diagnostic potential of the metabolite differences was investigated in this study. Glutamate, glycerol and choline had a high diagnostic accuracy; in contrast, isoleucine, leucine, pyruvate, lysine, α-ketoglutarate, betaine, glycine, creatine, serine, lactate, tyrosine, phenylalanine, histidine and formate enabled the accurate differentiation of BL mice from normal mice. The discovery of abnormal metabolism and relevant differential metabolites may provide useful clues for developing novel, noninvasive approaches for the diagnosis and prognosis of BL based on these potential biomarkers.

A copper-catalyzed domino reaction to construct functionalized indolizinones

An efficient copper-catalyzed domino reaction to 3,8a-disubstituted indolizinones has been firstly developed, and the protocol uses pyridine ketones and terminal alkynes as the starting materials, overriding the isolation of propargylic alcohols.

Selective recognition of c-myc promoter G-quadruplex and down-regulation of oncogene c-myc transcription in human cancer cells by 3,8a-disubstituted indolizinone

C-myc promoter G-quadruplex is a very important target for developing anti-cancer drugs. However, highly selective recognition of DNA c-myc G-quadruplex with parallel configuration is also a very challenging problem. Here, we showed a new type of N-containing alkaloid, 3,8a-disubstituted indolizinone, which adopted a distorted configuration. 6-Methyl-3-(naphthalen-2-yl)-8a-(4-methylpyridin-2-yl)-indolizinone could selectively recognize DNA c-myc G-quadruplex leading to an obvious fluorescence enhancement by 11-fold, and meanwhile the G-quadruplex can be stabilized up to 90 °C. Further, it could down-regulate the transcription of oncogene c-myc in both human non-small cell line (A549) and human laryngeal epithelial cell line (Hep-2).

G-quadruplex induced chirality of methylazacalix[6]pyridine via unprecedented binding stoichiometry: en route to multiplex controlled molecular switch

Nucleic acid based molecular device is a developing research field which attracts great interests in material for building machinelike nanodevices. G-quadruplex, as a new type of DNA secondary structures, can be harnessed to construct molecular device owing to its rich structural polymorphism. Herein, we developed a switching system based on G-quadruplexes and methylazacalix[6]pyridine (MACP6). The induced circular dichroism (CD) signal of MACP6 was used to monitor the switch controlled by temperature or pH value. Furthermore, the CD titration, Job-plot, variable temperature CD and 1H-NMR experiments not only confirmed the binding mode between MACP6 and G-quadruplex, but also explained the difference switching effect of MACP6 and various G-quadruplexes. The established strategy has the potential to be used as the chiral probe for specific G-quadruplex recognition.

Ethyl-substitutive Thioflavin T as a highly-specific fluorescence probe for detecting G-quadruplex structure

G-quadruplex has attracted considerable attention due to their prevalent distribution in functional genomic regions and transcripts, which can importantly influence biological processes such as regulation of telomere maintenance, gene transcription and gene translation. Artificial receptor study has been developed for accurate identification of G-quadruplex from DNA species, since it is important for the G-quadruplex related basic research, clinical diagnosis, and therapy. Herein, fluorescent dye ThT-E, a derivative of the known fluorescence probe Thioflavin T (ThT), was designed and synthesized to effectively differentiate various G-quadruplex structures from other nucleic acid forms. Compared with methyl groups in ThT, three ethyl groups were introduced to ThT-E, which leads to strengthened affinity, selectivity and little inducing effect on the G-quadruplex formation. More importantly, ThT-E could be served as a visual tool to directly differentiate G-quadruplex solution even with naked eyes under illumination of ultraviolet light. Thus, this probe reported herein may hold great promise for high-throughput assay to screen G-quadruplex, which may widely apply to G-quadruplex-based potential diagnosis and therapy.

Direct visualization of nucleolar G-quadruplexes in live cells by using a fluorescent light-up probe

BACKGROUND Direct detection of G-quadruplexes in human cells has become an important issue due to the vital role of G-quadruplex related to biological functions. Despite several probes have been developed for detection of the G-quadruplexes in cytoplasm or whole cells, the probe being used to monitor the nucleolar G-quadruplexes is still lacking. METHODS Formation of the nucleolar G-quadruplex structures was confirmed by using circular dichroism (CD) spectroscopy. The binding affinity and selectivity of Thioflavin T (ThT) towards various DNA/RNA motifs in solution and gel system were measured by using fluorescence spectroscopy and polyacrylamide gel electrophoresis (PAGE), respectively. G-quadruplex imaging in live cells was directly captured by using confocal laser scanning microscopy (CLSM). RESULTS Formation of the rDNA and rRNA G-quadruplex structures is demonstrated in vitro. ThT is found to show much higher affinity and selectivity towards these G-quadruplex structures versus other nucleic acid motifs either in solution or in gel system. The nucleolar G-quadruplexes in living cells are visualized by using ThT as a fluorescent probe. G-quadruplex-ligand treatments in live cells lead to sharp decrease of ThT signal. CONCLUSIONS The natural existence of the G-quadruplexes structure in the nucleoli of living cells is directly visualized by using ThT as an indicator. GENERAL SIGNIFICANCE The research provides substantive evidence for formation of the rRNA G-quadruplex structures, and also offers an effective probe for direct visualization of the nucleolar G-quadruplexes in living cells.