Copper is an essential trace element in living organisms with its levels and localisation being carefully managed by the cellular machinery. However, if misregulated, deficiency or excess of copper ions can lead to several diseases. Therefore, it is important to have reliable methods to detect, monitor and visualise this metal in cells. Herein we report a new optical probe based on BODIPY, which shows a switch-on in its fluorescence intensity upon binding to copper(I), but not in the presence of high concentration of other physiologically relevant metal ions. More interestingly, binding to copper(I) leads to significant changes in the fluorescence lifetime of the new probe, which can be used to visualize copper(I) pools in lysosomes of live cells via fluorescence lifetime imaging microscopy (FLIM).
Copper/analysis , Boron Compounds/chemistry , Boron Compounds/toxicity , Cell Line, Tumor , Copper/chemistry , Fluorescent Dyes/chemistry , Fluorescent Dyes/toxicity , Humans , Lysosomes/chemistry , Microscopy, Fluorescence/methods
Guanine rich regions of oligonucleotides fold into quadruple-stranded structures called G-quadruplexes (G4s). 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 G4s within nuclei of live and fixed cells. We present a FLIM-based cellular assay to study the interaction of non-fluorescent small molecules with G4s 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 G4s in these cells, implying that FancJ and RTEL1 play a role in resolving G4 structures in cellulo.
DNA/metabolism , G-Quadruplexes , Intravital Microscopy/methods , Molecular Imaging/methods , Animals , Cell Line, Tumor , DNA/chemistry , DNA Helicases/genetics , DNA Helicases/metabolism , Fanconi Anemia Complementation Group Proteins/genetics , Fanconi Anemia Complementation Group Proteins/metabolism , Fibroblasts , Fluorescent Dyes/chemistry , Gene Knockdown Techniques , Humans , Indoles/chemistry , Mice , Microscopy, Fluorescence/methods , RNA Helicases/genetics , RNA Helicases/metabolism
Four-stranded G-quadruplex (G4) DNA is a non-canonical DNA topology that has been proposed to form in cells and play key roles in how the genome is read and used by the cellular machinery. Previously, a fluorescent triangulenium probe (DAOTA-M2) was used to visualise G4s in cellulo, thanks to its distinct fluorescence lifetimes when bound to different DNA topologies. Herein, the library of available triangulenium probes is expanded to explore how modifications to the fluorescent core of the molecule affect its photophysical characteristics, interaction with DNA and cellular localisation. The benzo-bridged and isopropyl-bridged diazatriangulenium dyes, BDATA-M2 and CDATA-M2 respectively, featuring ethyl-morpholino substituents, were synthesised and characterised. The interactions of these molecules with different DNA topologies were studied to determine their binding affinity, fluorescence enhancement and fluorescence lifetime response. Finally, the cellular uptake and localisation of these optical probes were investigated. Whilst structural modifications to the triangulenium core only slightly alter the binding affinity to DNA, BDATA-M2 and CDATA-M2 cannot distinguish between DNA topologies through their fluorescence lifetime. It is argued theoretically and experimentally that this is due to reduced effectiveness of photoinduced electron transfer (PET) quenching. This work presents valuable new evidence into the critical role of PET quenching when using the fluorescence lifetime of triangulenium dyes to discriminate G4 DNA from duplex DNA, highlighting the importance of fine tuning redox and spectral properties when developing new triangulenium-based G4 probes.
DNA/analysis , DNA/chemistry , Fluorescence , Fluorescent Dyes/chemistry , G-Quadruplexes , Electron Transport , Fluorescent Dyes/analysis , Molecular Probes/analysis , Molecular Probes/chemistry
Many gram-positive bacteria produce bacillithiol to aid in the maintenance of redox homeostasis and degradation of toxic compounds, including the antibiotic fosfomycin. Bacillithiol is produced via a three-enzyme pathway that includes the action of the zinc-dependent deacetylase BshB. Previous studies identified conserved aspartate and histidine residues within the active site that are involved in metal binding and catalysis, but the enzymatic mechanism is not fully understood. Here we report two X-ray crystallographic structures of BshB from Bacillus subtilis that provide insight into the BshB catalytic mechanism.
Amidohydrolases/chemistry , Bacillus subtilis/enzymology , Bacterial Proteins/chemistry , Cysteine/analogs & derivatives , Glucosamine/analogs & derivatives , Zinc/metabolism , Amidohydrolases/metabolism , Bacterial Proteins/metabolism , Biocatalysis , Crystallography, X-Ray , Cysteine/biosynthesis , Cysteine/chemistry , Glucosamine/biosynthesis , Glucosamine/chemistry , Models, Molecular , Protein Conformation , Zinc/chemistry
Methylated bases in tRNA, rRNA and mRNA control a variety of cellular processes, including protein synthesis, antimicrobial resistance and gene expression. Currently, bulk methods that report the average methylation state of ~104-107 cells are used to detect these modifications, obscuring potentially important biological information. Here, we use in situ hybridization of Molecular Beacons for single-cell detection of three methylations (m62A, m1G and m3U) that destabilize Watson-Crick base pairs. Our method-methylation-sensitive RNA fluorescence in situ hybridization-detects single methylations of rRNA, quantifies antibiotic-resistant bacteria in mixtures of cells and simultaneously detects multiple methylations using multicolor fluorescence imaging.
In Situ Hybridization, Fluorescence/methods , RNA, Ribosomal/metabolism , RNA/metabolism , Single-Cell Analysis/methods , Adenine/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Guanine/metabolism , Methylation , Methyltransferases/genetics , Methyltransferases/metabolism , Microscopy, Fluorescence , RNA/genetics , RNA, Ribosomal/genetics , Uridine/metabolism
