Rong Huang

Mitochondrial DNA (mtDNA) is known to play a critical role in cellular functions. However, the fluorescent probe enantio-selectively targeting live-cell mtDNA is rare. We recently found that the well-known DNA ‘light-switch’ [Ru(phen)₂dppz]Cl₂ can image nuclear DNA in live-cells with chlorophenolic counter-anions via forming lipophilic ion-pairing complex. Interestingly, after washing with fresh-medium, [Ru(phen)₂dppz]Cl₂ was found to re-localize from nucleus to mitochondria via ABC transporter proteins. Intriguingly, the two enantiomers of [Ru(phen)₂dppz]Cl₂ were found to bind enantio-selectively with mtDNA in live-cells not only by super-resolution optical microscopy techniques (SIM, STED), but also by biochemical methods (mitochondrial membrane staining with Tomo20-dronpa). Using [Ru(phen)₂dppz]Cl₂ as the new mtDNA probe, we further found that each mitochondrion containing 1–8 mtDNA molecules are distributed throughout the entire mitochondrial matrix, and there are more nucleoids near nucleus. More interestingly, we found enantio-selective apoptotic cell death was induced by the two enantiomers by prolonged visible light irradiation, and in-situ self-monitoring apoptosis process can be achieved by using the unique ‘photo-triggered nuclear translocation’ property of the Ru complex. This is the first report on enantio-selective targeting and super-resolution imaging of live-cell mtDNA by a chiral Ru complex via formation and dissociation of ion-pairing complex with suitable counter-anions.

Targeted and enantioselective delivery of chiral diagnostic-probes and therapeutics into specific compartments inside cells is of utmost importance in the improvement of disease detection and treatment. The classical DNA ‘light-switch’ ruthenium(II)-polypyridyl complex, [Ru(DIP)₂(dppz)]Cl₂ (DIP = 4,7-diphenyl-1,10-phenanthroline, dppz = dipyridophenazine) has been shown to be accumulated only in the cytoplasm and membrane, but excluded from its intended nuclear DNA target. In this study, the cationic [Ru(DIP)₂(dppz)]²⁺ is found to be redirected into live-cell nucleus in the presence of lipophilic 3,5-dichlorophenolate or flufenamate counter-anions via ion-pairing mechanism, while maintaining its original DNA recognition characteristics. Interestingly and unexpectedly, further studies show that only the Δ-enantiomer is selectively translocated into nucleus while the Λ-enantiomer remains trapped in cytoplasm, which is found to be mainly due to their differential enantioselective binding affinities with cytoplasmic proteins and nuclear DNA. More importantly, only the nucleus-relocalized Δ-enantiomer can induce obvious DNA damage and cell apoptosis upon prolonged visible-light irradiation. Thus, the use of Δ-enantiomer can significantly reduce the dosage needed for maximal treatment effect. This represents the first report of enantioselective targeting and photosensitization of classical Ru(II) complex via simple ion-pairing with suitable weak acid counter-anions, which opens new opportunities for more effective enantioselective cancer treatment.

An in-depth understanding of the mechanisms of cellular uptake and efflux would facilitate the design of metal complexes with not only better functionality and targeted theranostic efficiency, but also with controlled toxicity. Here we find, unexpectedly, that the DNA “light-switching” Ru(II)-polypyridyl complex [Ru(phen)₂(dppz)]²⁺ already delivered to the nucleus via ion-pairing with chlorophenolate counter-anions can gradually efflux to the cytoplasm when the cells were washed and incubated with fresh culture-medium. Interestingly, [Ru(phen)₂(dppz)]²⁺ effluxed to the cytoplasm can be redirected back to the nucleus when the chlorophenolate counter-anions were added again. The efflux of nuclear [Ru(phen)₂(dppz)]²⁺ was found to be mediated mainly via ATP-binding cassette (ABC) transporter proteins. Analogous reversible, but enantio-selective nuclear uptake and efflux were observed with the two pure chiral forms of [Ru(phen)₂(dppz)]Cl₂. This represents the first report of reversible and controllable nuclear uptake and efflux of a DNA “light-switching” Ru(II)-complex in living-cells via ion-pairing, which should provide novel insights for future research on using ion-pairing as an effective approach to control the cellular uptake and redistribution of other potential theranostic metal complexes.

The high-resolution technique transmission electron microscopy (TEM), with OsO₄ as the traditional fixative, is an essential tool for cell biology and medicine. Although OsO₄ has been extensively used, it is far from perfect because of its high volatility and toxicity. Os(II) polypyridyl complexes like [Os(phen)₂(dppz)]²⁺ (phen = 1,10-phenanthroline; dppz = dipyridophenazine) are not only the well-known molecular DNA “light-switches” but also the potential ideal candidates for TEM studies. Here, we report that the cell-impermeable cationic [Os(phen)₂(dppz)]²⁺ can be preferentially delivered into the live-cell nucleus through ion-pairing with chlorophenolate counter-anions, where it functions as an unparalleled enantioselective nuclear DNA imaging reagent especially suitable for correlative light and electron microscopy (CLEM) studies in both living and fixed cells, which can clearly visualize chromosome aggregation and decondensation during mitosis simultaneously. We propose that the chiral Os(II) polypyridyl complexes can be used as a distinctive group of enantioselective high-resolution CLEM imaging probes for live-cell nuclear DNA studies.

We have found recently that nuclear uptake of the cell-impermeable DNA light-switching Ru(II)-polypyridyl cationic complexes such as [Ru(bpy)₂(dppz)]Cl₂ was remarkably enhanced by pentachlorophenol (PCP), by forming ion-pairing complexes via a passive diffusion mechanism. However, it is not clear whether the enhanced nuclear uptake of [Ru(bpy)₂(dppz)]²⁺ is only limited to PCP, or it is a general phenomenon for other highly chlorinated phenols (HCPs); and if so, what are the major physicochemical factors in determining nuclear uptake? Here, we found that the nuclear uptake of [Ru(bpy)₂(dppz)]²⁺ can also be facilitated by other two groups of HCPs including three tetrachlorophenol (TeCP) and six trichlorophenol (TCP) isomers. Interestingly and unexpectedly, 2,3,4,5-TeCP was found to be the most effective one for nuclear delivery of [Ru(bpy)₂(dppz)]²⁺, which is even better than the most-highly chlorinated PCP, and much better than its two other TeCP isomers. Further studies showed that the nuclear uptake of [Ru(bpy)₂(dppz)]²⁺ was positively correlated with the binding stability, but to our surprise, inversely correlated with the lipophilicity of the ion-pairing complexes formed between [Ru(bpy)₂(dppz)]Cl₂ and HCPs. These findings should provide new perspectives for future investigations on using ion-pairing as an effective method for delivering other bio-active metal complexes into their intended cellular targets.

The nucleolus is an important subnuclear structure, but very few dyes are available for nucleolar imaging. Here we show that the Λ-enantiomer of [Os(phen)₂(dppz)]Cl₂ can differentially distinguish the nucleolus from nucleus in living cells with tetrachlorophenolate as counteranion, while the Δ-enantiomer can do so in fixed cells by FLIM imaging. Further studies with three specific metabolic inhibitors for nucleolar protein synthesis found that the lifetime changes of the two enantiomers in the nucleolus can reflect the alteration of the cellular microenvironment, which is related to the general pathological status of the nucleolus. We then observed dynamical architecture changes of the nucleolus, chromosome and spindle apparatus during cell differentiation by these two enantiomers. The chiral Os(II) complex shows many advantages as compared to the commercially available nucleolus dye Syto 9: it displays a much larger Stokes shift value with a near-red emission and a longer lifetime, it can image spindle apparatus during mitosis, and more importantly, it is enantioselective.

Delivering potential theranostic metal complexes into preferential cellular targets is becoming of increasing interest. Here we report that nuclear uptake of a cell-impermeable DNA “light-switching” Ru(II)-polypyridyl complex can be significantly facilitated by chlorophenolate counter-anions, which was found, unexpectedly, to be correlated positively with the binding stability but inversely with the lipophilicity of the formed ion pairs.

Dehairing alkaline protease (DHAP) from Bacillus pumilus BA06 has been demonstrated to have high catalytic efficiency and good thermostability, with potential application in leather processing. In order to get insights into its catalytic mechanism, two mutants with single amino acid substitution according to the homology modeling and multiple sequence alignment were characterized in thermodynamics of thermal denaturation and temperature dependence of substrate hydrolysis. The results showed that both mutants of V149I and R249E have a systematic increase in catalytic efficiency (k_cat/K_m) in a wide range of temperatures, mainly due to an increase of k₁ (substrate diffusion) and k₂ (acylation) for V149I and of k₂ and k₃ (deacylation) for R249E. In comparison with the wild-type DHAP, the thermostability is increased for V149I and decreased for R249E. Thermodynamic analysis indicated that the free energy (ΔG_a°) of activation for thermal denaturation may govern the thermostability. The value of ΔG_a° is increased for V149I and decreased for R249E. Based on these data and the structural modeling, it is suggested that substitution of Val149 with Ile may disturb the local flexibility in the substrate-binding pocket, leading to enhancement of binding affinity for the substrate. In contrast, substitution of Arg249 with Glu leads to interruption of interaction with the C-terminal of enzyme, thus resulting in less thermostability. This study indicates that amino acid residues in the active center or in the substrate-binding pocket may disturb the catalytic process and can be selected as the target for protein engineering in the bacterial alkaline proteases.