Yuqi Chen

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.

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.

Fluorescent Strategy Based on Cationic Conjugated Polymer Fluorescence Resonance Energy Transfer for the Quantification of 5-(Hydroxymethyl)cytosine in Genomic DNA

DNA methylation is dynamically reprogrammed during early embryonic development in mammals. It can be explained partially by the discovery of 5-(hydroxymethyl)cytosine (5-hmC), 5-formylcytosine (5-fC), and 5-carboxylcytosine (5-caC), which are identified as key players involved in both active and passive demethylation pathways. As one of the ten-eleven translocation oxidation products, 5-hmC was found relatively abundant in neuron cells and embryonic stem cells. Herein we report a new method for 5-hmC quantification in genomic DNA based on CCP-FRET (cationic conjugated polymers act as the energy donor and induce fluorescence resonance energy transfer) assay combined with KRuO4 oxidation. 5-hmC in genomic DNA can be selectively transformed into 5-fC by the oxidation of KRuO4 and then labeled with hydroxylamine-BODIPY (BODIPY = 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) fluorophore through the reaction between 5-fC and hydroxylamine-BODIPY. After the fluorescently labeled DNA was captured by CCP through electrostatic interactions, a significant FRET between CCP and hydroxylamine-BODIPY fluorophore was observed. This CCP-FRET-based assay benefits from light-harvesting, large Stokes shift, and optical signal amplification properties of the CCP. Furthermore, this CCP-FRET-based assay was quite successfully demonstrated for the 5-hmC quantification in three types of cells (mESc, HeLa, HEK 293T), providing a much more convenient choice for 5-hmC quantification in genomic DNA.

Nonlinear optical dye TSQ1 as an efficiently selective fluorescent probe for G-quadruplex DNA

A squarylium dye TSQ1 shows a remarkable fluorescence enhancement selectivity for the G-quadruplex DNA structure against duplex DNA, it can be distinguished even by the naked eye. What is more important, TSQ1 did not induce the G-rich sequence folding into G-quadruplex structure which means TSQ1 can be used to detect G-quadruplex structures in cells.

Discrimination between 5-hydroxymethylcytosine and 5-methylcytosine in DNA by selective chemical labeling

Detection of DNA damage has been greatly improved following the development of equipment and techniques, however, discrimination between 5-hydroxymethylcytosine (5-hmC) and 5-methylcytosine (5-mC) is still a thorny problem. In the present study, an approach to oxidize and selective label (Ox-Labeling) 5-hmC in native DNA has been reported, which conveniently distinguishes 5-hmC from 5-mC using simple and effective processes.

A highly efficient fluorescence-based switch-on detection method of 5-formyluracil in DNA

The identification of hydroxylmethyl- and formylpyrimidines in genomic DNA was a landmark event in epigenetics. Numerous laboratories in related fields are investigating the biology of these and other nucleic acid modifications. However, limitations in the ability to detect and synthesize appropriate modifications are an impediment. Herein, we explored a remarkable development in the selective detection of 5-formyluracil in both single-stranded and double-stranded DNA under mild conditions. The “switch-on” specificity towards 5-formyluracil enabled a high signal-to-noise ratio in qualitatively and quantitatively detecting materials containing 5-formyluracil, which is not affected by the presence of abasic sites and 5-formylcytosine, the modified cytosine counterpart of 5-formyluracil. In summary, the innoxiousness, convenience, and cost-efficiency of the 5-formyluracil phosphoramidite synthetic routine would promote the understanding of the epigenetic role of this natural thymidine modification.

Fluorescent turn-on probes for the detection of fluoride ions in organic solvent and in cells

We have successfully developed two coumarin-based fluorescent probes to detect F−in vitro. Probe 1 is lipophilic for organic soluted detection. Probe 2 is an amphipathic molecule due to its TBDPS and triphenyl-phosphine groups for both mitochondrial localization and fluoride ion detection. Both of the two probes have good selectivity in F− detection; the detection limit of probe 2 was as low as 670 nM. Probe 2 has good fluorescence enhancement in response to F− in cell imaging. The probes are lowly cytotoxic to cells.

A novel aggregation-induced emission fluorescent probe for nucleic acid detection and its applications in cell imaging.

A new kind of aggregation-induced emission compound was synthesized and used as the probe of nucleic acid. The characterization of this compound was studied. Both the RNA and DNA were detected by using this probe. And the detection scope of DNA and RNA was different. We researched the selectivity of our probe in double and single strand DNA sequences. The visualization of gel electrophoresis and the cell nucleus imaging were researched as well. Compared with the traditional nucleus dye Hoechst 33258, our probe also has the potential to be nucleus dye. And the cell toxicity was well performed by MTT assays.

pH-controlled DNAzymes: Rational design and their applications in DNA-machinery devices

The availability and reliability of strategies for molecular biosensing over a finely adjustable dynamic range is essential to enhance the understanding and control of vital biological process. To expand the versatility and utility of nucleic acidrelated enzymes, we demonstrated a rational approach to acquiring tunable, pH-dependent deoxyribozymes (DNAzymes) with catalytic activities and response sensitivities that can be tuned through a simple change in solution pH. To do this, we capitalized upon the pH dependence of Hoogsteen interactions and designed i-motif- and triplex-based DNAzymes that can be finely regulated with high precision over a physiologically relevant pH interval. The modified DNAzymes are dependent upon pH for efficient cleavage of substrates, and their catalytic performance can be tuned by regulating the sequence of inserted i-motif/triplex structures. The principle of tunable, pH-dependent DNAzymes provides the opportunity to engineer pH-controlled DNA-machinery devices with unprecedented sensitivity to pH changes. For example, we constructed a DNA-walker device, the stepping rate of which could be adjusted by simply modulating solution pH within an interval of 5.6 to 7.4, as well as a DNA tetrahedron that can be opened at pH 6.4 and kept closed at pH 7.4. The potential of this approach is not limited to serve as pH-dependent devices, but rather may be combined with other elements to expand their practical usefulness.

An L-DNA G-Quadruplex: Application for Peroxidase DNAzyme

L-DNA is the mirror-image form of natural D-DNA. We demonstrate that one left-handed G-rich sequence can form an L-DNA intramolecular G-quadruplex. Further investigation revealed that a DNAzyme formed by an L-nucleotide G-quadruplex exhibited peroxidase catalytic efficiency. The enhancement of the color change of the oxygenation product ABTS•− caused by L-nucleotide G-quadruplex formation could be clearly observed with naked eyes. This research provides a new concept for the application of the L-DNA peroxidase DNAzyme complex in nuclease-containing biological systems.