Pu Guo

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.

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.

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.

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.

A mitochondria-targeted zinc(II) phthalocyanine for photodynamic therapy

A water-soluble zinc(II) phthalocyanine (ZnPc1) substituted with 1-(3-methyl) imidazoliumylethyloxy on the periphery was synthesized, and its subcellular localization and photodynamic activities were studied. ZnPc1 exhibited an almost exclusive mitochondrial-localizing property in human cervical carcinoma (HeLa) cells. Remarkable photocytotoxicity but low dark cytotoxic properties were observed for ZnPc1 in four different cancer cell lines. Photodynamic treatment (PDT) with ZnPc1 resulted in the generation of reactive oxygen species, a collapse of the mitochondrial membrane potential and chromatin condensation, all important hallmarks of apoptosis. The results are consistent with its mitochondrial localization property and qualify ZnPc1 as a promising PDT agent.

A selective turn-on fluorescence strategy for the detection of 5-hydroxymethyl-2′-deoxycytidine

A new type of 1,3-O,N-heterocycle, including two fluorescent derivatives, has been synthesised by the reaction of the hydroxyl and amine groups of 5-hydroxymethyl-2′-deoxycytidine (hmC). This reactivity is shown to enable the selective recognition of hmC over other natural nucleotides in aqueous buffer.