Research Projects

Mitochondrial DNA (mtDNA) of mammalian cells is about 16,569 base pairs long, and it contains a high amount of guanine (G) bases, especially on the heavy (H) strand, which tend to form four-stranded DNA structures called G-quadruplexes (G4s). Mitochondrial G4s (mtG4s) are associated with various diseases like cancer and neurodegenerative disorders. mtG4s regulate mitochondrial replication and transcription by blocking the enzymes needed for these processes; therefore, they are being explored as potential therapeutic targets for various diseases. Despite the biological significance of mtG4s, their detection in live cells remains challenging due to their structural dynamics and accessibility. Recently, a limited number of fluorescent probes have been developed to target mtG4s, but only a few have shown satisfactory light-up properties inside the cells. The current challenges, such as poor mitochondrial permeability, selectivity towards mtG4s, weak fluorescence signal, photo-bleaching, off-target light up, and high toxicity in live cells, demand the need to develop fluorescent probes for mtG4 cellular imaging. Herein, we propose to develop novel and red emissive mtG4-sensing probes that rely on (Photo-electron transfer) PET and ICT (Intramolecular charge transfer) phenomena, containing a donor-π-acceptor module along with mitochondrial targeting components: triphenylphosphine (TPP), guanidine, or mitochondrion penetrating peptide (mpp). These probes are expected to show a large Stokes shift as they have extended  conjugation, which will help the probe to produce bright fluorescent signals. The fluorescent probes will be based on benzoxadiazole, 1,2-diaminobenzonitrile, and furanylidenepropanedinitrile scaffolds. (Figure 1, in methodology) Our preliminary studies showed that this red-emissive probe (BnQ), based on benzoxadiazole, lights up mtG4 structures in live HeLa cells. 1,2-Diaminobenzonitrile-based probes coupled with coumarin and piperidine derivatives (CBn and PBn) are expected to show twisted-intramolecular charge transfer when interacting with G4 DNAs. Further, furanylidenepropanedinitrile-based probes (IPF) contain three cyanide groups acting as strong electron acceptors to provide high brightness in live cells upon target recognition. These design elements will help to achieve target selectivity inside the cell, absorption in the far-red region, fluorescence in the NIR region, and minimal interference from the cellular environment. These probes will be optimized to have low toxicity for safe real-time imaging of mtG4s and help to unravel their biological functions. After the synthesis of the probes is carried out by straightforward synthetic strategies, biophysical studies such as fluorescence titration, TCSPC analysis, quantum yield calculation, and CD-based experiments will be utilized to unravel the interaction of the probes with various mtG4s and duplex DNAs. Further, the fluorescence gel staining assay will be performed to study the fluorescence behavior of the probes with mtG4s. The toxicity of the probes in cancerous cells and non-cancerous cells (HeLa, HepG2, MCF-7, and HEK293T) will be checked, and cell imaging studies will be carried out to visualize the mtG4s in live and fixed cells using confocal and super-resolution microscopy. To confirm the localization of mitochondria in live cells, co-localization experiments with mitotracker green will be performed, followed by DNase and RNase digestion experiments to confirm the interaction of probes with DNA or RNA. The results from these studies are expected to expand our understanding of mitochondrial function during the cell cycle and uncover how mtG4s influence gene expression and cellular metabolism. They could also open up new tools and strategies for real-time visualization of mitochondrial genome dynamics, thereby opening avenues to develop mtG4-targeted therapeutics for cancer and neglected neurodegenerative diseases.