Tingting Hong

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.

Ag+ and cysteine detection by Ag+–guanine interaction based on graphene oxide and G-quadruplex DNA

Sensitive and selective techniques for both Ag+ and Cys detection have been designed based on graphene oxide (GO) and G-quadruplex DNA. The G-quadruplex structure can capture the fluorescent molecule acridine orange (AO) from the surface of graphene oxide (GO), which will recover the fluorescence of AO that was initially quenched by GO. Then the addition of Ag+ will release AO back to GO, because Ag+ can destroy the G-quadruplex structure, giving a fluorescence quenching process again. Based on this “turn off” principle, a highly sensitive and selective Ag+ detection method was developed. In addition, cysteine (Cys) can capture Ag+ from the guanine base of DNA which will result in the regeneration of the G-quadruplex DNA structure and thus the regeneration of the fluorescence signal. This “turn on” principle can be used for the development of a highly selective and sensitive detection method for Cys.

Fluorescein Derivatives as Bifunctional Molecules for the Simultaneous Inhibiting and Labeling of FTO Protein

The FTO protein is unequivocally reported to play a critical role in human obesity and in the regulation of cellular levels of m(6)A modification, which makes FTO a significant and worthy subject of study. Here, we identified that fluorescein derivatives can selectively inhibit FTO demethylation, and the mechanisms behind these activities were elucidated after we determined the X-ray crystal structures of FTO/fluorescein and FTO/5-aminofluorescein. Furthermore, these inhibitors can also be applied to the direct labeling and enrichment of FTO protein combined with photoaffinity labeling assay.

Highly Selective Detection of 5-Methylcytosine in Genomic DNA Based on Asymmetric PCR and Specific DNA Damaging Reagents

DNA methylation is a significant epigenetic modification of the genome that is involved in regulating many cellular processes. An increasing number of human diseases have been discovered to be associated with aberrant DNA methylation, and aberrant DNA methylation has been deemed to be a potential biomarker for diseases such as cancers. A safe, nontoxic, and sensitive method for accurate detection of 5-methylcytosine in genomic DNA is extremely useful for early diagnosis and therapy of cancers. In this paper, we established a novel system to detect 5-methylcytosine, which is based on bisulfite treatment, asymmetric PCR, and specific DNA damaging reagents. Our method could be used for identifying the loci of 5mC in genomic DNA and detecting the DNA methylation levels in tissues as well.

Luminescence Sensing for Qualitative and Quantitative Detection of 5-Methylcytosine

5-Methylcytosine (5mC) is revealed as a heritable epigenetic modification in genomic DNA. It has been reported that cytosine/guanine dinucleotides (CpG) hypermethylation in the promoter regions of tumor suppressor genes is related with inappropriate gene silencing, so the determination of 5mC in the CpG islands of mammals has attracted much attention. In this paper, a luminescence sensing strategy based on bisulfite treatment, asymmetric polymerase chain reaction (PCR), and adenosine triphosphate (ATP)-releasing nucleotide is proposed. With little background, this method can provide accurate quantitative information about methylation changes at CpG sites, even at a specific site. The proposed method can be successfully employed to determine the methylation status of three hepatocellular carcinomas (HCC) related genes in clinical tissues.

Application of Ammonium Persulfate for Selective Oxidation of Guanines for Nucleic Acid Sequencing

Nucleic acids can be sequenced by a chemical procedure that partially damages the nucleotide positions at their base repetition. Many methods have been reported for the selective recognition of guanine. The accurate identification of guanine in both single and double regions of DNA and RNA remains a challenging task. Herein, we present a new, non-toxic and simple method for the selective recognition of guanine in both DNA and RNA sequences via ammonium persulfate modification. This strategy can be further successfully applied to the detection of 5-methylcytosine by using PCR.