N-Methylmesoporphyrin IX Fluorescence As A Reporter Of Strand Orientation In Guanine Quadruplexes

Guanine quadruplexes (GQ) are four-stranded DNA structures formed by guanine-rich DNA sequences. The formation of GQs inhibits cancer cell growth, although the detection of GQs invivo has proven difficult, in part because of their structural diversity. The development of GQ-selective fluorescent reporters would enhance our ability to quantify the number and location of GQs, ultimately advancing biological studies of quadruplex relevance and function. N-methylmesoporphyrin IX (NMM) interacts selectively with parallel-stranded GQs; in addition, its fluorescence is sensitive to the presence of DNA, making this ligand a possible candidate for a quadruplex probe. In the present study, we investigated the effect of DNA secondary structure on NMM fluorescence. We found that NMM fluorescence increases by about 60-fold in the presence of parallel-stranded GQs and by about 40-fold in the presence of hybrid GQs. Antiparallel GQs lead to lower than 10-fold increases in NMM fluorescence. Single-stranded DNA, duplex, or i-motif, induce no change in NMM fluorescence. We conclude that NMM shows promise as a turn-on\u27 fluorescent probe for detecting quadruplex structures, as well as for differentiating them on the basis of strand orientation

G-Quadruplex Dynamics Contribute To Regulation Of Mitochondrial Gene Expression

Single-stranded DNA or RNA sequences rich in guanine (G) can adopt non-canonical structures known as G-quadruplexes (G4). Mitochondrial DNA (mtDNA) sequences that are predicted to form G4 are enriched on the heavy-strand and have been associated with formation of deletion breakpoints. Increasing evidence supports the ability of mtDNA to form G4 in cancer cells; however, the functional roles of G4 structures in regulating mitochondrial nucleic acid homeostasis in non-cancerous cells remain unclear. Here, we demonstrate by live cell imaging that the G4-ligand RHPS4 localizes primarily to mitochondria at low doses. We find that low doses of RHPS4 do not induce a nuclear DNA damage response but do cause an acute inhibition of mitochondrial transcript elongation, leading to respiratory complex depletion. We also observe that RHPS4 interferes with mtDNA levels or synthesis both in cells and isolated mitochondria. Importantly, a mtDNA variant that increases G4 stability and anti-parallel G4-forming character shows a stronger respiratory defect in response to RHPS4, supporting the conclusion that mitochondrial sensitivity to RHPS4 is G4-mediated. Taken together, our results indicate a direct role for G4 perturbation in mitochondrial genome replication, transcription processivity, and respiratory function in normal cells

Interactions Of Ruthenium(II) Polypyridyl Complexes With Human Telomeric DNA

Eight [Ru(bpy)₂L]²⁺ and three [Ru(phen)₂L]²⁺ complexes (where bpy = 2,2′-bipyridine and phen = 1,10-phenanthroline are ancillary ligands, and L = a polypyridyl experimental ligand) were investigated for their G-quadruplex binding abilities. Fluorescence resonance energy transfer melting assays were used to screen these complexes for their ability to selectively stabilize human telomeric DNA variant, Tel22. The best G-quadruplex stabilizers were further characterized for their binding properties (binding constant and stoichiometry) using UV–vis, fluorescence spectroscopy, and mass spectrometry. The ligands\u27 ability to alter the structure of Tel22 was determined via circular dichroism and PAGE studies. We identified me₂allox as the experimental ligand capable of conferring excellent stabilizing ability and good selectivity to polypyridyl Ru(II) complexes. Replacing bpy by phen did not significantly impact interactions with Tel22, suggesting that binding involves mostly the experimental ligand. However, using a particular ancillary ligand can help fine-tune G-quadruplex-binding properties of Ru(II) complexes. Finally, the fluorescence “light switch” behavior of all Ru(II) complexes in the presence of Tel22 G-quadruplex was explored. All Ru(II) complexes displayed “light switch” properties, especially [Ru(bpy)₂(diamino)]²⁺, [Ru(bpy)₂(dppz)]²⁺, and [Ru(bpy)₂(aap)]2²⁺ Current work sheds light on how Ru(II) polypyridyl complexes interact with human telomeric DNA with possible application in cancer therapy or G-quadruplex sensing

G-Quadruplexes: A Role In The Mitochondrial Genome Stability

Single-stranded DNA or RNA regions rich in guanine (G) sequences can adopt non-canonical G-quadruplexes (G4) structures through the formation of Hoogsteen hydrogen bonds. Several studies report the existence of G4 structure formation both in vitro and in vivo, and have established their biological importance in nuclear DNA replication, transcriptional regulation and genome stability [1,2]. Genomic events, such as replication, lead to single-strand DNA formation and increase the probability of G4 formation, which could contribute to genome instability both in nuclear and mitochondrial DNA (mtDNA). The mitochondrial genome is present in thousands of copies per cell as a double-stranded circular molecule of 16 kb, encoding 13 proteins essential to oxidative phosphorylation (OXPHOS) and the RNAs necessary for their translation. Our recent in vitro study established that mtDNA has the potential to form G4 structures [3]. The same study demonstrated a tight correlation between G4 motifs and mtDNA deletion breakpoints, supporting a role for the G-quadruplexes in genome instability. To better understand the biological function of G-quadruplexes in mitochondria, we screened G4 stabilizing ligands for effects on mtDNA abundance. Here we report the activity a specific mitochondrial GQ ligand on mtDNA stability, gene expression and mitochondrial respiration