Content-aware frame interpolation (CAFI): deep learning-based temporal super-resolution for fast bioimaging.

The development of high-resolution microscopes has made it possible to investigate cellular processes in 3D and over time. However, observing fast cellular dynamics remains challenging because of photobleaching and phototoxicity. Here we report the implementation of two content-aware frame interpolation (CAFI) deep learning networks, Zooming SlowMo and Depth-Aware Video Frame Interpolation, that are highly suited for accurately predicting images in between image pairs, therefore improving the temporal resolution of image series post-acquisition. We show that CAFI is capable of understanding the motion context of biological structures and can perform better than standard interpolation methods. We benchmark CAFI's performance on 12 different datasets, obtained from four different microscopy modalities, and demonstrate its capabilities for single-particle tracking and nuclear segmentation. CAFI potentially allows for reduced light exposure and phototoxicity on the sample for improved long-term live-cell imaging. The models and the training and testing data are available via the ZeroCostDL4Mic platform.

Cellular Visualization of G-Quadruplex RNA via Fluorescence- Lifetime Imaging Microscopy.

Over the past decade, appreciation of the roles of G-quadruplex (G4) structures in cellular regulation and maintenance has rapidly grown, making the establishment of robust methods to visualize G4s increasingly important. Fluorescent probes are commonly used for G4 detection in vitro; however, achieving sufficient selectivity to detect G4s in a dense and structurally diverse cellular environment is challenging. The use of fluorescent probes for G4 detection is further complicated by variations of probe uptake into cells, which may affect fluorescence intensity independently of G4 abundance. In this work, we report an alternative small-molecule approach to visualize G4s that does not rely on fluorescence intensity switch-on and, thus, does not require the use of molecules with exclusive G4 binding selectivity. Specifically, we have developed a novel thiazole orange derivative, TOR-G4, that exhibits a unique fluorescence lifetime when bound to G4s compared to other structures, allowing G4 binding to be sensitively distinguished from non-G4 binding, independent of the local probe concentration. Furthermore, TOR-G4 primarily colocalizes with RNA in the cytoplasm and nucleoli of cells, making it the first lifetime-based probe validated for exploring the emerging roles of RNA G4s in cellulo.

A Semi-Automated, High-Throughput Approach for the Synthesis and Identification of Highly Photo-Cytotoxic Iridium Complexes.

The discovery of new compounds with pharmacological properties is usually a lengthy, laborious and expensive process. Thus, there is increasing interest in developing workflows that allow for the rapid synthesis and evaluation of libraries of compounds with the aim of identifying leads for further drug development. Herein, we apply combinatorial synthesis to build a library of 90 iridium(III) complexes (81 of which are new) over two synthesise-and-test cycles, with the aim of identifying potential agents for photodynamic therapy. We demonstrate the power of this approach by identifying highly active complexes that are well-tolerated in the dark but display very low nM phototoxicity against cancer cells. To build a detailed structure-activity relationship for this class of compounds we have used density functional theory (DFT) calculations to determine some key electronic parameters and study correlations with the experimental data. Finally, we present an optimised semi-automated synthesise-and-test protocol to obtain multiplex data within 72 hours.

Dimeric Metal-Salphen Complexes Which Target Multimeric G-Quadruplex DNA.

G-Quadruplex DNA structures have attracted increasing attention due to their biological roles and potential as targets for the development of new drugs. While most guanine-rich sequences in the genome have the potential to form monomeric G-quadruplexes, certain sequences have enough guanine-tracks to give rise to multimeric quadruplexes. One of these sequences is the human telomere where tandem repeats of TTAGGG can lead to the formation of two or more adjacent G-quadruplexes. Herein we report on the modular synthesis via click chemistry of dimeric metal-salphen complexes (with NiII and PtII) bridged by either polyether or peptide linkers. We show by circular dichroism (CD) spectroscopy that they generally have higher selectivity for dimeric vs monomeric G-quadruplexes. The emissive properties of the PtII-salphen dimeric complexes have been used to study their interactions with monomeric and dimeric G-quadruplexes in vitro as well as to study their cellular uptake and localization.

Platinum(II)-Based Optical Probes for Imaging Quadruplex DNA Structures via Phosphorescence Lifetime Imaging Microscopy.

G-quadruplex DNA is a non-canonical structure that forms in guanine-rich regions of the genome. There is increasing evidence showing that G-quadruplexes have important biological functions, and therefore molecular tools to visualise these structures are important. Herein we report on a series of new cyclometallated platinum(II) complexes which, upon binding to G-quadruplex DNA, display an increase in their phosphorescence, acting as switch-on probes. More importantly, upon binding to G-quadruplexes they display a selective and distinct lengthening of their emission lifetime. We show that this effect can be used to selectively visualise these structures in cells using Phosphorescence Lifetime Imaging Microscopy (PLIM).

Radiolabelling Pt-based quadruplex DNA binders via click chemistry.

Guanine-rich sequences of DNA and RNA can fold into intramolecular tetra-helical assemblies known as G-quadruplexes (G4). Their formation in vivo has been associated to a range of biological functions and therefore they have been identified as potential drug targets. Consequently, a broad range of small molecules have been developed to target G4s. Amongst those are metal complexes with Schiff base ligands. Herein, we report the functionalisation of one of these well-established G4 DNA binders (based on a square planar platinum(II)-salphen complex) with two different radiolabelled complexes. An 111In-conjugate was successfully used to assess its in vivo distribution in a mouse tumour model using single-photon emission computed tomography (SPECT) imaging. These studies highlighted the accumulation of this Pt-salphen-111In conjugate in the tumour.

Assessing The Key Photophysical Properties of Triangulenium Dyes for DNA Binding by Alteration of the Fluorescent Core.

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.

Rotaxanes as Cages to Control DNA Binding, Cytotoxicity, and Cellular Uptake of a Small Molecule*.

The efficacy of many drugs can be limited by undesirable properties, such as poor aqueous solubility, low bioavailability, and "off-target" interactions. To combat this, various drug carriers have been investigated to enhance the pharmacological profile of therapeutic agents. In this work, we demonstrate the use of mechanical protection to "cage" a DNA-targeting metallodrug within a photodegradable rotaxane. More specifically, we report the synthesis of rotaxanes incorporating as a stoppering unit a known G-quadruplex DNA binder, namely a PtII -salphen complex. This compound cannot interact with DNA when it is part of the mechanically interlocked assembly. The second rotaxane stopper can be cleaved by either light or an esterase, releasing the PtII -salphen complex. This system shows enhanced cell permeability and limited cytotoxicity within osteosarcoma cells compared to the free drug. Light activation leads to a dramatic increase in cytotoxicity, arising from the translocation of PtII -salphen to the nucleus and its binding to DNA.

Selective Detection of Cu+ Ions in Live Cells via Fluorescence Lifetime Imaging Microscopy.

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).

An Electroactive Oligo-EDOT Platform for Neural Tissue Engineering.

The unique electrochemical properties of the conductive polymer poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) make it an attractive material for use in neural tissue engineering applications. However, inadequate mechanical properties, and difficulties in processing and lack of biodegradability have hindered progress in this field. Here, the functionality of PEDOT:PSS for neural tissue engineering is improved by incorporating 3,4-ethylenedioxythiophene (EDOT) oligomers, synthesized using a novel end-capping strategy, into block co-polymers. By exploiting end-functionalized oligoEDOT constructs as macroinitiators for the polymerization of poly(caprolactone), a block co-polymer is produced that is electroactive, processable, and bio-compatible. By combining these properties, electroactive fibrous mats are produced for neuronal culture via solution electrospinning and melt electrospinning writing. Importantly, it is also shown that neurite length and branching of neural stem cells can be enhanced on the materials under electrical stimulation, demonstrating the promise of these scaffolds for neural tissue engineering.

Development of Polyamine-Substituted Triphenylamine Ligands with High Affinity and Selectivity for G-Quadruplex DNA.

Currently, significant efforts are devoted to designing small molecules able to bind selectively to guanine quadruplexes (G4s). These noncanonical DNA structures are implicated in various important biological processes and have been identified as potential targets for drug development. Previously, a series of triphenylamine (TPA)-based compounds, including macrocyclic polyamines, that displayed high affinity towards G4 DNA were reported. Following this initial work, herein a series of second-generation compounds, in which the central TPA has been functionalised with flexible and adaptive linear polyamines, are presented with the aim of maximising the selectivity towards G4 DNA. The acid-base properties of the new derivatives have been studied by means of potentiometric titrations, UV/Vis and fluorescence emission spectroscopy. The interaction with G4s and duplex DNA has been explored by using FRET melting assays, fluorescence spectroscopy and circular dichroism. Compared with previous TPA derivatives with macrocyclic substituents, the new ligands reported herein retain the G4 affinity, but display two orders of magnitude higher selectivity for G4 versus duplex DNA; this is most likely due to the ability of the linear substituents to embrace the G4 structure.

An Octahedral Cobalt(III) Complex with Axial NH3 Ligands that Templates and Selectively Stabilises G-quadruplex DNA.

Guanine-rich sequences of DNA are known to readily fold into tetra-stranded helical structures known as G-quadruplexes (G4). Due to their biological relevance, G4s are potential anticancer drug targets and therefore there is significant interest in molecules with high affinity for these structures. Most G4 binders are polyaromatic planar compounds which π-π stack on the G4's guanine tetrad. However, many of these compounds are not very selective since they can also intercalate into duplex DNA. Herein we report a new class of binder based on an octahedral cobalt(III) complex that binds to G4 via a different mode involving hydrogen bonding, electrostatic interactions and π-π stacking. We show that this new compound binds selectivity to G4 over duplex DNA (particularly to the G-rich sequence of the c-myc promoter). This new octahedral complex also has the ability to template the formation of G4 DNA from the unfolded sequence. Finally, we show that upon binding to G4, the complex prevents helicase Pif1-p from unfolding the c-myc G4 structure.

Binding Studies of Metal-Salphen and Metal-Bipyridine Complexes towards G-Quadruplex DNA.

The proposed in vivo formation of G-quadruplex DNA (G4 DNA) in promoter regions of oncogenes and in telomeres has prompted the development of small molecules with high affinity and selectivity for these structures. Herein we report the synthesis of a new di-substituted bipyridine ligand and the corresponding complexes with Ni2+ and VO2+ . Both these new complexes have been characterized spectroscopically and by X-ray crystallography. Detailed DNA binding studies of these two complexes, together with three previously reported metal salphen complexes, are presented. Using FRET melting assays, the binding affinity and selectivity of the five metal complexes against six different G4 DNA structures as well as a duplex DNA have been determined. In addition, we present detailed ITC and UV/Vis studies to characterize the interaction of the complexes with human telomeric G4 DNA. Finally, we show via a polymerase stop assay that these complexes are able to stabilize a G4 DNA structure (from the c-Myc oncogene promoter) and halt the activity of Taq polymerase.

Aza-Macrocyclic Triphenylamine Ligands for G-Quadruplex Recognition.

A new series of triphenylamine-based ligands with one (TPA1PY), two (TPA2PY) or three pendant aza-macrocycle(s) (TPA3PY) has been synthesised and studied by means of pH-metric titrations, UV/Vis spectroscopy and fluorescence experiments. The affinity of these ligands for G-quadruplex (G4) DNA and the selectivity they show for G4s over duplex DNA were investigated by Förster resonance energy transfer (FRET) melting assays, fluorimetric titrations and circular dichroism spectroscopy. Interestingly, the interactions of the bi- and especially the tri-branched ligands with G4s lead to a very intense redshifted fluorescence emission band that may be associated with intermolecular aggregation between the molecule and DNA. This light-up effect allows the application of the ligands as fluorescence probes to selectively detect G4s.

A Redox-Activated G-Quadruplex DNA Binder Based on a Platinum(IV)-Salphen Complex.

There has been increasing interest in the development of small molecules that can selectively bind to G-quadruplex DNA structures. The latter have been associated with a number of key biological processes and therefore are proposed to be potential targets for drug development. Herein, we report the first example of a reduction-activated G-quadruplex DNA binder. We show that a new octahedral platinum(IV)-salphen complex does not interact with DNA in aqueous media at pH 7.4; however, upon addition of bioreductants such as ascorbic acid or glutathione, the compound is readily reduced to the corresponding square planar platinum(II) complex. In contrast to the parent platinum(IV) complex, the in situ generated platinum(II) complex has good affinity for G-quadruplex DNA.

Dinickel-Salphen Complexes as Binders of Human Telomeric Dimeric G-Quadruplexes.

Three new polyether-tethered dinickel-salphen complexes (2 a-c) have been synthesized and fully characterized by NMR spectroscopy, mass spectrometry, and elemental analyses. The binding affinity and selectivity of these complexes and of the parent mono-nickel complex (1) towards dimeric quadruplex DNA have been determined by UV/Vis titrations, fluorescence spectroscopy, CD spectroscopy, and electrophoresis. These studies have shown that the dinickel-salphen complex with the longest polyether linker (2 c) has higher binding affinity and selectivity towards dimeric quadruplexes (over monomeric quadruplexes) than the dinickel-salphen complexes with the shorter polyether linkers (2 a and 2 b). Complex 2 c also has higher selectivity towards human telomeric dimeric quadruplexes with one TTA linker than the monometallic complex 1. Based on the spectroscopic data, a possible binding mode between complex 2 c and the dimeric G-quadruplex DNA under study is proposed.

Experimental Determination of Zinc Isotope Fractionation in Complexes with the Phytosiderophore 2'-Deoxymugeneic Acid (DMA) and Its Structural Analogues, and Implications for Plant Uptake Mechanisms.

The stable isotope signatures of zinc and other metals are increasingly used to study plant and soil processes. Complexation with phytosiderophores is a key reaction and understanding the controls of isotope fractionation is central to such studies. Here, we investigated isotope fractionation during complexation of Zn2+ with the phytosiderophore 2'-deoxymugeneic acid (DMA), and with three commercially available structural analogues of DMA: EDTA, TmDTA, and CyDTA. We used ion exchange chromatography to separate free and complexed zinc, and identified appropriate cation exchange resins for the individual systems. These were Chelex-100 for EDTA and CyDTA, Amberlite CG50 for TmDTA and Amberlite IR120 for DMA. With all the ligands we found preferential partitioning of isotopically heavy zinc in the complexed form, and the extent of fractionation was independent of the Zn:ligand ratio used, indicating isotopic equilibrium and that the results were not significantly affected by artifacts during separation. The fractionations (in ‰) were +0.33 ± 0.07 (1σ, n = 3), + 0.45 ± 0.02 (1σ, n = 2), + 0.62 ± 0.05 (1σ, n = 3) and +0.30 ± 0.07 (1σ, n = 4) for EDTA, TmDTA, CyDTA, and DMA, respectively. Despite the similarity in Zn-coordinating donor groups, the fractionation factors are significantly different and extent of fractionation seems proportional to the complexation stability constant. The extent of fractionation with DMA agreed with observed fractionations in zinc uptake by paddy rice in field experiments, supporting the possible involvement of DMA in zinc uptake by rice.

Trianguleniums as Optical Probes for G-Quadruplexes: A Photophysical, Electrochemical, and Computational Study.

Nucleic acids can adopt non-duplex topologies, such as G-quadruplexes in vitro. Yet it has been challenging to establish their existence and function in vivo due to a lack of suitable tools. Recently, we identified the triangulenium compound DAOTA-M2 as a unique fluorescence probe for such studies. This probe's emission lifetime is highly dependent on the topology of the DNA it interacts with opening up the possibility of carrying out live-cell imaging studies. Herein, we describe the origin of its fluorescence selectivity for G-quadruplexes. Cyclic voltammetry predicts that the appended morpholino groups can act as intra- molecular photo-induced electron transfer (PET) quenchers. Photophysical studies show that a delicate balance between this effect and inter-molecular PET with nucleobases is key to the overall fluorescence enhancement observed upon nucleic acid binding. We utilised computational modelling to demonstrate a conformational dependence of intra-molecular PET. Finally, we performed orthogonal studies with a triangulenium compound, in which the morpholino groups were removed, and demonstrated that this change inverts triangulenium fluorescence selectivity from G-quadruplex to duplex DNA, thus highlighting the importance of fine tuning the molecular structure not only for target affinity, but also for fluorescence response.

Quadruplex DNA-Stabilising Dinuclear Platinum(II) Terpyridine Complexes with Flexible Linkers.

Four dinuclear terpyridineplatinum(II) (Pt-terpy) complexes were investigated for interactions with G-quadruplex DNA (QDNA) and duplex DNA (dsDNA) by synchrotron radiation circular dichroism (SRCD), fluorescent intercalator displacement (FID) assays and fluorescence resonance energy transfer (FRET) melting studies. Additionally, computational docking studies were undertaken to provide insight into potential binding modes for these complexes. The complexes demonstrated the ability to increase the melting temperature of various QDNA motifs by up to 17 °C and maintain this in up to a 600-fold excess of dsDNA. This study demonstrates that dinuclear Pt-terpy complexes stabilise QDNA and have a high degree of selectivity for QDNA over dsDNA.

NMR Structure of a Triangulenium-Based Long-Lived Fluorescence Probe Bound to a G-Quadruplex.

An NMR structural study of the interaction between a small-molecule optical probe (DAOTA-M2) and a G-quadruplex from the promoter region of the c-myc oncogene revealed that they interact at 1:2 binding stoichiometry. NMR-restrained structural calculations show that binding of DAOTA-M2 occurs mainly through π-π stacking between the polyaromatic core of the ligand and guanine residues of the outer G-quartets. Interestingly, the binding affinities of DAOTA-M2 differ by a factor of two for the outer G-quartets of the unimolecular parallel G-quadruplex under study. Unrestrained MD calculations indicate that DAOTA-M2 displays significant dynamic behavior when stacked on a G-quartet plane. These studies provide molecular guidelines for the design of triangulenium derivatives that can be used as optical probes for G-quadruplexes.