Unprecedented enantio-selective live-cell mitochondrial DNA super-resolution imaging and photo-sensitizing by the chiral ruthenium polypyridyl DNA “light-switch”

Abstract

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.

Probing a Major DNA Weakness: Resolving the Groove and Sequence Selectivity of the Diimine Complex Λ‐[Ru(phen)2phi]2+

“…In that context, recent papers show great progress, encompassing diruthenium compounds, compounds for photodynamic therapy, and the use of counteranions to facilitate cellular takeup. [34][35][36][37][38][39] In contrast, studies of DNA binding by the diimine complex [Ru(phen) 2 phi] 2 + have a longer history than that of the dppz complexes, dating back to the 1980s, but with binding modes much less well understood. This complex was first synthesised as part of the early work on mixed ligand complexes of ruthenium binding to DNA.…”

Shining New Light on Biological Systems: Luminescent Transition Metal Complexes for Bioimaging and Biosensing Applications

“…887 High-resolution imaging with SIM and STED microscopy shows that [Ru(phen) 2 (dppz)] 2+ localizes at the center of the mitochondrion and intercalates into mitochondrial DNA, revealing that each mitochondrion contains 1 to 8 DNA molecules distributed throughout the entire mitochondrial matrix. 888 Furthermore, the complex can induce enantioselective apoptotic cell death upon continuous visiblelight irradiation, with the Δ-isomer showing higher photocytotoxicity due to its strong binding with mitochondrial DNA. Notably, the complex is translocated from the mitochondria back to the nucleus during apoptotic cell death, offering a direct method for in situ self-monitoring of cell apoptosis.…”

“…Structure–property relationship analysis reveals that a high binding affinity between the cationic complex and the counteranion, as well as low lipophilicity of the resultant ion-pair complex, are both important to minimize nonspecific interactions with hydrophobic proteins and membranes in the cytoplasm of the cells, facilitating the nuclear enrichment of the complexes. , However, upon incubation in a fresh culture medium, the complexes are effluxed out of the cell nucleus by the ATP-binding cassette transporters and relocated to the mitochondria . High-resolution imaging with SIM and STED microscopy shows that [Ru(phen) 2 (dppz)] 2+ localizes at the center of the mitochondrion and intercalates into mitochondrial DNA, revealing that each mitochondrion contains 1 to 8 DNA molecules distributed throughout the entire mitochondrial matrix . Furthermore, the complex can induce enantioselective apoptotic cell death upon continuous visible-light irradiation, with the Δ-isomer showing higher photocytotoxicity due to its strong binding with mitochondrial DNA.…”