Biology subjects

Edel, J. B.

Publications and source records attributed to Edel, J. B..

3 recordsLinked to original sources

bioRxiv2025

Assessing PARP trapping dynamics in ovarian cancer using a CRISPR-engineered FRET biosensor

Poly(ADP-ribose) polymerase inhibitors (PARPi) have revolutionised the treatment of ovarian high grade serous carcinoma (HGSC), especially those with defective homologous recombination. However, the emergence of resistance poses a critical challenge, as over 50% of patients relapse within three years. The mechanisms underlying changes in PARP trapping, a central aspect of PARPi efficacy, are not well understood due to limitations in current experimental methodologies. Existing techniques lack resolution and throughput, impeding efforts to study PARP trapping dynamics over time with single-cell resolution. Effective tools to study PARP trapping in live cells are urgently needed to elucidate resistance mechanisms and inform therapeutic strategies. To address this, we used CRISPR-Cas9 gene editing to dual-label endogenous PARP1 with EGFP and mCherryFP in OVCAR4 cells to develop a novel intramolecular FRET-based biosensor that enables real-time, single-cell visualization of PARP trapping dynamics in live cells. High-content fluorescence lifetime imaging microscopy (FLIM) revealed dose dependent PARP trapping upon exposure to PARP inhibitor and distinguished between the trapping efficiencies of four different PARPi (veliparib, olaparib, rucaparib, talazoparib). Moreover, we found reduced PARP trapping in PARPi-resistant models, both in vitro and in vivo, providing critical evidence for altered PARP trapping as a resistance mechanism and illustrating the potential of this FRET biosensor to interrogate resistance mechanisms quantitatively. This PARP trapping biosensor represents a transformative advance, enabling dynamic, high-resolution analysis of mechanisms underlying cancer drug resistance. It provides critical insights into the heterogeneity of PARPi resistance, with implications for developing more effective therapies and improving personalised treatment strategies for ovarian cancer patients. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=58 SRC="FIGDIR/small/642798v1_ufig1.gif" ALT="Figure 1"> View larger version (11K): org.highwire.dtl.DTLVardef@1c5ee67org.highwire.dtl.DTLVardef@1a6d101org.highwire.dtl.DTLVardef@e3a2bborg.highwire.dtl.DTLVardef@d0ba03_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2020

Visualising G-quadruplex DNA dynamics in live cells by fluorescence lifetime imaging microscopy

Guanine rich regions of oligonucleotides fold into quadruple-stranded structures called G-quadruplexes (G4). Increasing evidence suggests that these G4 structures form in vivo and play a crucial role in cellular processes. However, their direct observation in live cells remains a challenge. Here we demonstrate that a fluorescent probe (DAOTA-M2) in conjunction with Fluorescence Lifetime Imaging Microscopy (FLIM) can identify G4 within nuclei of live and fixed cells. We present a new FLIM-based cellular assay to study the interaction of non-fluorescent small molecules with G4 and apply it to a wide range of drug candidates. We also demonstrate that DAOTA-M2 can be used to study G4 stability in live cells. Reduction of FancJ and RTEL1 expression in mammalian cells increases the DAOTA-M2 lifetime and therefore suggests an increased number of G4 in these cells, implying that FancJ and RTEL1 play a role in resolving G4 structures in cellulo.

biophysics↗

bioRxiv2019

Single-molecule nanopore sensing of actin dynamics and drug binding

Actin is a key protein in the dynamic processes within the eukaryotic cell. To date, methods exploring the molecular state of actin are limited to insights gained from structural approaches, providing a snapshot of protein folding, or methods that require chemical modifications compromising actin monomer thermostability. Nanopore sensing permits label-free investigation of native proteins and is ideally suited to study proteins such as actin that require specialised buffers and cofactors. Using nanopores we determined the state of actin at the macromolecular level (filamentous or globular) and in its monomeric form bound to inhibitors. We revealed urea-dependent and voltage-dependent transitional states and observed unfolding process within which sub-populations of transient actin oligomers are visible. We detected, in real-time, drug-binding and filament-growth events at the single-molecule level. This enabled us to calculate binding stoichiometries and to propose a model for protein dynamics using unmodified, native actin molecules, demostrating the promise of nanopores sensing for in-depth understanding of protein folding landscapes and for drug discovery.

biophysics↗