Ta-Chau Chang

Investigation of spectral conversion of d(TTAGGG)4 and d(TTAGGG)13 upon potassium titration by a G-quadruplex recognizer BMVC molecule.

We have introduced a G-quadruplex-binding ligand, 3,6-bis(1-methyl-4-vinylpyridinium)carbazole diiodide (BMVC), to verify the major structure of d(T2AG3)4 (H24) in potassium solution and examine the structural conversion of H24 in sodium solution upon potassium titration. The studies of circular dichroism, induced circular dichroism, spectral titration and gel competition have allowed us to determine the binding mode and binding ratio of BMVC to the H24 in solution and eliminate the parallel form as the major G-quadruplex structure. Although the mixed-type form could not be eliminated as a main component, the basket and chair forms are more likely the main components of H24 in potassium solution. In addition, the circular dichroism spectra and the job plots reveal that a longer telomeric sequence d(T2AG3)13 (H78) could form two units of G4 structure both in sodium or potassium solutions. Of particular interest is that no appreciable change on the induced circular dichroism spectra of BMVC is found during the change of the circular dichroism patterns of H24 upon potassium titration. Considering similar spectral conversion detected for H24 and a long sequence H78 together with the G4 structure stabilized by BMVC, it is therefore unlikely that the rapid spectral conversion of H24 and H78 is due to structural change between different types of the G4 structures. With reference to the circular dichroism spectra of d(GAA)7 and d(GAAA)5, we suggest that the spectral conversion of H24 upon potassium titration is attributed to fast ion exchange resulting in different loop base interaction and various hydrogen bonding effects.

Selective photodynamic therapy based on aggregation-induced emission enhancement of fluorescent organic nanoparticles

Three binary molecule conjugates were designed and synthesized by conjugating a chromophore (3, 6-bis-(1-methyl-4-vinylpyridinium)-carbazole diiodide, BMVC) to mono-, bis- and trishydroxyl photosensitizers, respectively. BMVC plays the role of cancer cells recognizer; AIEE (aggregation-induced emission enhancement) generator and FRET (Fluorescence Resonance Energy Transfer) donor. The self assembling properties of these binary conjugates result in different degrees of AIEE and then achieve the formations of FONs (fluorescent organic nanoparticles), which present efficient FRET and singlet oxygen generations. Biologically, FONs-photosensitizers from these compounds were much more phototoxicities to cancer cell than to normal cell without significant dark toxicity. In addition, their intracellular fluorescent colors switching upon photo-excitation are expected to be used for further cell death biomarker applications. This improved photodynamic activity might be due to the aggregation of compounds in the cell that form FONs which can promote PDT (photodynamic therapy) and are observed in cancer cell but not normal cell.

G-Quadruplex Stabilizer 3,6-Bis(1-Methyl-4-Vinylpyridinium)Carbazole Diiodide Induces Accelerated Senescence and Inhibits Tumorigenic Properties in Cancer Cells

Carbazole derivatives that stabilized G-quadruplex DNA structure formed by human telomeric sequence have been designed and synthesized. Among them, 3,6-bis(1-methyl-4-vinylpyridinium)carbazole diiodide (BMVC) showed an increase in G-quadruplex melting temperature by 13°C and has a potent inhibitory effect on telomerase activity. Treatment of H1299 cancer cells with 0.5 μmol/L BMVC did not cause acute toxicity and affect DNA replication; however, the BMVC-treated cells ceased to divide after a lag period. Hallmarks of senescence, including morphologic changes, detection of senescence-associated β-galactosidase activity, and decreased bromodeoxyuridine incorporation, were detected in BMVC-treated cancer cells. The BMVC-induced senescence phenotype is accompanied by progressive telomere shortening and detection of the DNA damage foci, indicating that BMVC caused telomere uncapping after long-term treatments. Unlike other telomerase inhibitors, the BMVC-treated cancer cells showed a fast telomere shortening rate and a lag period of growth before entering senescence. Interestingly, BMVC also suppressed the tumor-related properties of cancer cells, including cell migration, colony-forming ability, and anchorage-independent growth, indicating that the cellular effects of BMVC were not limited to telomeres. Consistent with the observations from cellular experiments, the tumorigenic potential of cancer cells was also reduced in mouse xenografts after BMVC treatments. Thus, BMVC repressed tumor progression through both telomere-dependent and telomere-independent pathways. (Mol Cancer Res 2008;6(6):955–64)

Direct evidence of mitochondrial G-quadruplex DNA by using fluorescent anti-cancer agents

G-quadruplex (G4) is a promising target for anti-cancer treatment. In this paper, we provide the first evidence supporting the presence of G4 in the mitochondrial DNA (mtDNA) of live cells. The molecular engineering of a fluorescent G4 ligand, 3,6-bis(1-methyl-4-vinylpyridinium) carbazole diiodide (BMVC), can change its major cellular localization from the nucleus to the mitochondria in cancer cells, while remaining primarily in the cytoplasm of normal cells. A number of BMVC derivatives with sufficient mitochondrial uptake can induce cancer cell death without damaging normal cells. Fluorescence studies of these anti-cancer agents in live cells and in isolated mitochondria from HeLa cells have demonstrated that their major target is mtDNA. In this study, we use fluorescence lifetime imaging microscopy to verify the existence of mtDNA G4s in live cells. Bioactivity studies indicate that interactions between these anti-cancer agents and mtDNA G4 can suppress mitochondrial gene expression. This work underlines the importance of fluorescence in the monitoring of drug-target interactions in cells and illustrates the emerging development of drugs in which mtDNA G4 is the primary target.

Fluorescent probe for visualizing guanine-quadruplex DNA by fluorescence lifetime imaging microscopy

Abstract. The importance of guanine-quadruplex (G4) is not only in protecting the ends of chromosomes for human telomeres but also in regulating gene expression for several gene promoters. However, the existence of G4 structures in living cells is still in debate. A fluorescent probe, 3,6-bis(1-methyl-2-vinylpyridinium) carbazole diiodide (o-BMVC), for differentiating G4 structures from duplexes is characterized. o-BMVC has a large contrast in fluorescence decay time, binding affinity, and fluorescent intensity between G4 structures and duplexes, which makes it a good candidate for probing G4 DNA structures. The fluorescence decay time of o-BMVC upon interaction with G4 structures of telomeric G-rich sequences is ∼2.8  ns and that of interaction with the duplex structure of a calf thymus is ∼1.2  ns. By analyzing its fluorescence decay time and histogram, we were able to detect one G4 out of 1000 duplexes in vitro. Furthermore, by using fluorescence lifetime imaging microscopy, we demonstrated an innovative methodology for visualizing the localization of G4 structures as well as mapping the localization of different G4 structures in living cells.

Isolation, purification and characterization of hemerythrin from Methylococcus capsulatus (Bath)

Earlier work from our laboratory has indicated that a hemerythrin-like protein was over-produced together with the particulate methane monooxygenase (pMMO) when Methylococcus capsulatus (Bath) was grown under high copper concentrations. A homologue of hemerythrin had not previously been found in any prokaryote. To confirm its identity as a hemerythrin, we have isolated and purified this protein by ion-exchange, gel-filtration and hydrophobic interaction chromatography, and characterized it by mass spectrometry, UV-visible, CD, EPR and resonance Raman spectroscopy. On the basis of biophysical and multiple sequence alignment analysis, the protein isolated from M. capsulatus (Bath) is in accord with hemerythrins previously reported from higher organisms. Determination of the Fe content in conjunction with molecular-weight estimation and mass analysis indicates that the native hemerythrin in M. capsulatus (Bath) is a monomer with molecular mass 14.8 kDa, in contrast to hemerythrins from other eukaryotic organisms, where they typically exist as a tetramer or higher oligomers.

Structural basis of sodium–potassium exchange of a human telomeric DNA quadruplex without topological conversion

Understanding the mechanism of Na+/K+-dependent spectral conversion of human telomeric G-quadruplex (G4) sequences has been limited not only because of the structural polymorphism but also the lack of sufficient structural information at different stages along the conversion process for one given oligonucleotide. In this work, we have determined the topology of the Na+ form of Tel23 G4, which is the same hybrid form as the K+ form of Tel23 G4 despite the distinct spectral patterns in their respective nuclear magnetic resonance (NMR) and circular dichroism spectra. The spectral difference, particularly the well-resolved imino proton NMR signals, allows us to monitor the structural conversion from Na+ form to K+ form during Na+/K+ exchange. Time-resolved NMR experiments of hydrogen–deuterium exchange and hybridization clearly exclude involvement of the global unfolding for the fast Na+/K+ spectral conversion. In addition, the K+ titration monitored by NMR reveals that the Na+/K+ exchange in Tel23 G4 is a two-step process. The addition of K+ significantly stabilizes the unfolding kinetics of Tel23 G4. These results offer a possible explanation of rapid spectral conversion of Na+/K+ exchange and insight into the mechanism of Na+/K+ structural conversion in human telomeric G4s.

Aggregation induced photodynamic therapy enhancement based on linear and nonlinear excited FRET of fluorescent organic nanoparticles

A binary molecule can self-assemble to form fluorescent organic nanoparticles (FONs) based on the Aggregation-Induced Emission Enhancement (AIEE) property and subsequently, presents an efficient fluorescence resonance energy transfer (FRET) to generate singlet oxygen under linear and nonlinear light sources. Biologically, this FON-photosensitizer is much more phototoxic to cancer cells than to normal cells without significant dark toxicity. Eventually, a new approach, called FON FRET-PDT or AIEE FRET-PDT, to promote the PDT effect is expected.

In-cell optical imaging of exogenous G-quadruplex DNA by fluorogenic ligands

Guanine-rich oligonucleotides (GROs) are promising therapeutic candidate for cancer treatment and other biomedical application. We have introduced a G-quadruplex (G4) ligand, 3,6-bis(1-methyl-4-vinylpyridinium) carbazole diiodide, to monitor the cellular uptake of naked GROs and map their intracellular localizations in living cells by using confocal microscopy. The GROs that form parallel G4 structures, such as PU22, T40214 and AS1411, are detected mainly in the lysosome of CL1-0 lung cancer cells after incubation for 2 h. On the contrary, the GROs that form non-parallel G4 structures, such as human telomeres (HT23) and thrombin binding aptamer (TBA), are rarely detected in the lysosome, but found mainly in the mitochondria. Moreover, the fluorescence resonant energy transfer studies of fluorophore-labeled GROs show that the parallel G4 structures can be retained in CL1-0 cells, whereas the non-parallel G4 structures are likely distorted in CL1-0 cells after cellular uptake. Of interest is that the distorted G4 structure of HT23 from the non-parallel G4 structure can reform to a probable parallel G4 structure induced by a G4 ligand in CL1-0 living cells. These findings are valuable to the design and rationale behind the possible targeted drug delivery to specific cellular organelles using GROs.

Label-free imaging of Drosophila in vivo by coherent anti-Stokes Raman scattering and two-photon excitation autofluorescence microscopy

Drosophila is one of the most valuable model organisms for studying genetics and developmental biology. The fat body in Drosophila, which is analogous to the liver and adipose tissue in human, stores lipids that act as an energy source during its development. At the early stages of metamorphosis, the fat body remodeling occurs involving the dissociation of the fat body into individual fat cells. Here we introduce a combination of coherent anti-Stokes Raman scattering (CARS) and two-photon excitation autofluorescence (TPE-F) microscopy to achieve label-free imaging of Drosophila in vivo at larval and pupal stages. The strong CARS signal from lipids allows direct imaging of the larval fat body and pupal fat cells. In addition, the use of TPE-F microscopy allows the observation of other internal organs in the larva and autofluorescent globules in fat cells. During the dissociation of the fat body, the findings of the degradation of lipid droplets and an increase in autofluorescent globules indicate the consumption of lipids and the recruitment of proteins in fat cells. Through in vivo imaging and direct monitoring, CARS microscopy may help elucidate how metamorphosis is regulated and study the lipid metabolism in Drosophila.