Separase is a key cysteine protease to separate sister chromatids through digestion of cohesion ring that inhibits chromosome segregation as trigger of metaphase–anaphase transition in eukaryotes and highly regulated its activity by binding with securin and cyclinB-CDK1 complex. Those bindings prevent proteolytic activity of separase until the onset of anaphase. Chromosome missegregation and aneuploidy are frequently observed in malignancies. However, there are some difficulties for biochemical examinations due to instability of separase in vitro and a few spatiotemporal resolution approaches monitoring live separase activity throughout mitotic processes. Here we have developed FRET-based molecular sensors including GFP variants with separase cleavable sequences as donor and covalently attached fluorescent dye as acceptor molecules applicable to conventional live cell imaging and flow cytometric analysis because of efficient live cell uptake. We investigated performance of equivalent molecular sensors localized or not localized inside nucleus under cell cycle control using flow cytometry. Synchronized cell cycle progression rendered significant separase activity detections in both molecular sensors. We obtained consistent outcomes with localized molecular sensor introduction and cell cycle control by fluorescent microscopic observations. We thus established live cell separase activity monitoring systems specifically or statistically that could lead to elucidate separase properties in detail.

Sub-micron o/w emulsions coated with chitosan have been used for drug delivery, quorum sensing inhibition and vaccine development. To study interactions with biological systems, nanocapsules have been fluorescently labelled in previous works, but it is often difficult to distinguish the released label from intact nanocapsules. In this study, we present advanced labelling strategies based on FRET measurements for chitosan-coated nanocapsules and investigate their dissolution and degradation. We used FRET measurements of nanocapsules loaded with equimolar concentrations of two fluorescent dyes in their oily core and correlated them with DLS count rate measurement and absorbance measurements during their disintegration by dissolution. Using count rate measurements, we also investigated the enzymatic degradation of nanocapsules using pancreatin and how protein corona formation influences their degradation. Of note, nanocapsules dissolved in ethanol, where FRET decreased simultaneously with count rate, and absorbance caused by nanocapsule turbidity, indicating increased distance between dye molecules after their release. Nanocapsules were degradable by pancreatin in a dose dependent manner, and showed a delayed enzymatic degradation after protein corona formation. We present here novel labelling strategies for nanocapsules that allow us to judge their status and an in vitro method to study nanocapsule degradation and the influence of surface characteristics.

Chitosan and its derivatives as a biocompatible, biodegradable polymer that has been well-established in biotechnology and medicine as the nanogels and nanofilms forming material, has a number of practical applications in the field of the supply of medicines, food colloids, agrotechnology, as well. In this paper, the focus of attention is directed to the study of the physico-chemical principles of the formation of gels and nano-particles using fluorescence spectroscopy methods. We have obtained chitosan (Chit5) and pegylated chitosan (Chit5-PEG) particles with an average size of 170 or 400 nm, respectively by counterflow extrusion method. The paper presents three based fluorescent probes sensitive to the formation of chitosan particles: congo red, malachite green as well as covalently attached pyrene-label. Dyes fluoresce about an order of magnitude brighter in the composition of chitosan nanogel particles or in mscrogels. With these fluorescence probes, the pH- and temperature-dependent behavior of polymers was studied, and the influence of the genipin -based crosslinking agent on the gel formation parameters, such as kinetics and concentration dependence of the nanoparticles formation accompanied by sharply increases in the fluorescence intensity of dyes was evaluated. A sensitive approach for tracking gelation and including of small drug molecules and protein by FRET using a pyrene-tryptophan donor-acceptor pair is proposed using tryptophan or ovalbumin as fluorophores-donors. Upon nanogel formation FRET effect is observed manifested in sharp increase if the fluorescence intensity of the pyrene (acceptor). Thus, the application of the fluorescent probes with FRET function for monitoring the gelation and nanoparticles formation as well as drug encapsulation in chitosan-based parts, which have broad prospects in biomedical practice and food industry.

Amyloid β-peptide (Aβ) oligomerization is believed to contribute to the neuronal dysfunction in Alzheimer disease (AD). Despite decades of research, many details of Aβ oligomerization in neurons still need to be revealed. Förster Resonance Energy Transfer (FRET) is a simple but effective way to study molecular interactions. Here we use a confocal microscope with a sensitive Airyscan detector for FRET detection. By live cell FRET imaging, we detect Aβ42 oligomerization in primary neurons. The neurons were incubated with fluorescently labelled Aβ42 in the cell culture medium for 24 hours. Aβ42 were internalized and oligomerized into the lysosomes/late endosomes in a concentration-dependent manner. Both the cellular uptake and intracellular oligomerization of Aβ42 were significantly higher than for Aβ40. These findings provide a better understanding of Aβ42 oligomerization in neurons.

Modern medical needs call for the efficient remedies against bacterial diseases including severe pulmonary infections reported as a secondary SARS-Cov-2 infection. Here, we propose imprinted drug ciprofloxacin (CF) into the polymer based on methyl-β-cyclodextrin (MCD) via template synthesis. The obtained polycarbamide nanoparticles possess CF's sustained drug release. The interaction of human serum albumin (HSA) with CF and CF-MCD carriers was conducted by FRET, FTIR, fluorescence, and circular dichroism spectroscopy. These studies uncovered that MCD decreases CF's binding efficiency by 2 times, whereas CF’s encapsulation in polymer matrix doubles the Ka value. The changes in HSA’s secondary structure indicate no alterations in the main mechanism of complex formation between CF and HSA in the presence of MCD-based carries. The drug delivery system demonstrates prolonged CF release at pH 7.4 in presence of HSA (model conditions of blood plasma). CF-MCD carriers inhibit E. coli and B. subtilis growth, but for MAX systems we observed a MIC’s increase (~2 times). We believe that our findings are important for further development of new efficient drug forms.

Fluorescent labels, especially FRET probes, are promising tools for studying a number of bio-chemical processes. In this paper, we promoted 2 important applications of FRET probes (MUTMAC-R6G and FITC-R6G): i) the formation of micelles from surfactants of various natures and polymers (chitosan – fatty acid), as well as ii) monitoring of enzymatic activity with improved parameters (increased analytical signal and improved selectivity due to shift to the long-wavelength region). The formation of micelles is accompanied by the convergence of fluorophores in the hydrophobic micelle core by a distance closer than in the buffer solution, thus r/R0 (where R0 – Foerster radius) is chosen as an analytical signal of micelle formation, including critical micelle concentration (CMC) and critical pre-micelle concentration (CPMC). The CMC values calculated using FRET probes are in a good agreement with literature data. At the same time, the r/R0 function provides valuable information about the nature and mechanism of micelle formation. With the second analytical application of FRET probes, we considered the optimization of techniques for studying enzymatic activity. The enzyme catalyses the reaction with the release of a fluorescent product, the signal from which may not be enough for detection, or it may be quenched, for example, in the reverse micelles (AOT-octane). Here we have proposed a solution – a FRET probe containing a rhodamine 6G (R6G) acceptor, which allows us to monitor the enzymatic reaction selectively (in the red region, 550-600 nm), and obtain a significantly higher fluorescence yield (potentially from 10 to 250 times). Thus, we have demonstrated a high potential for the FRET probes application as indicators of micelle formation as well as for the study of the enzyme catalytic activity. In the future, the method developed has prospects for application in the visualization of the enzyme functioning in cells due to the shift of the fluorescence signal to the long-wavelength region with an increase in the signal selectivity to suppress autofluorescence.

Silk fibroin is a well-known biopolymer used in several applications in which the interaction with biological tissue is required. In fact, fibroin is extremely versatile and can be shaped to form several constructs useful in tissue engineering applications. Confocal imaging is usually per-formed to test the cells behaviour on the construct and in this context the fibroin autofluorescence is regarded as a problem. In addition, the autofluorescence is not intense enough to provide useful morphological images. In fact, to control study the constructs morphology other techniques are used (i.e. SEM, Micro-CT). In this work we propose a method based on the fluorescence energy transfer (FRET) to suppress the fibroin autofluorescence moving it to higher wavelength accessible to the confocal microscopy for a direct imaging.

Proliferating cell nuclear antigen (PCNA) is the key regulator of human DNA metabolism. One important interaction partner is p15, involved in DNA replication and repair. Targeting the PCNA-p15 interaction is a promising therapeutic strategy against cancer. Here, a Förster resonance energy transfer (FRET)-based assay for the analysis of PCNA-p15 interaction was developed. Next to the application as screening tool for the identification and characterization of PCNA-p15 interaction inhibitors, the assay is also suitable for the investigation of mutation-induced changes in their affinity. This is particularly useful for analysing disease associated PCNA or p15 variants at the molecular level. Recently, the PCNA variant C148S has been associated with Ataxia-telangiectasia-like disorder type 2 (ATLD2). ATLD2 is a neurodegenerative disease based on defects in DNA repair due to an impaired PCNA. Incubation time dependent FRET measurements indicated no effect on PCNAC148S-p15 affinity, but on PCNA stability. The impaired stability and increased aggregation behaviour of PCNAC148S was confirmed by intrinsic tryptophan fluorescence, DSF and AF4 measurements. The analysis of the disease associated PCNA variant demonstrated the versatility of the interaction assay as developed.

NPM interaction with tumor suppressor p53 is a part of a complex interaction network and considerably affects cellular stress response. An impact of characteristic AML-associated NPM mutations on interaction with p53 has not been investigated yet, although consequences of NPMmut-induced p53 export to the cytoplasm are important for understanding of leukemogenic potential of these mutations. We investigated p53-NPM interaction in live HEK-293T cells by FLIM-FRET and in cell lysates by immunoprecipitation. Results were confirmed in leukemia cell lines. eGFP lifetime-photoconversion was used to follow redistribution dynamics of NPMmut and p53 in Selinexor-treated cells. We confirmed the p53-NPMwt interaction in intact cells and newly documented that this interaction is not compromised by the NPM mutation causing displacement of p53 to the cytoplasm. Importantly, the interaction was not abolished for non-oligomerizing NPM variants with truncated oligomerization domain, suggesting that oligomerization is not essential for interaction of NPM forms with p53. Inhibition of the nuclear exporter XPO1 by Selinexor caused expected nuclear relocalization of both NPMmut and p53. However, significantly different return rates of these proteins indicate nontrivial mechanism of p53 and NPMmut cellular trafficking. We suggest that the altered p53 regulation in cells expressing NPMmut offers a new target for AML therapy.

In this article/review, the selective interactions of several berberine and palmatine derivatives with various DNA G-quadruplex structures are reported. These derivatives were constructed starting from two natural compounds, berberine and palamatine, through specific synthetic passages following two different schemes for each of them and using several substituents. The details of these synthesis are also described. Indeed, the study of the interactions of these derivative compounds with various G-quadruplex forming sequences was carried out by means of various structural and biochemical techniques. The results show that the presence of suitable side chains are very useful to improve the interaction of the ligands with G-quadruplex structures. Thus, since G-quadruplex formation is promoted by these compounds, which have never been reported before, these may be tested as potential anticancer drugs.

The PII protein is an evolutionary highly conserved regulatory protein from bacteria to higher plants. In bacteria it modulates the activity of several enzymes, transporters and regulatory factors by interacting with them and thereby regulating important metabolic hubs like carbon/nitrogen homeostasis. More than two decades ago the PII protein was characterized for the first time in plants, but its physiological role is still not sufficiently resolved. To gain more insights into the function of this protein, we investigated the interaction behaviour of AtPII with candidate proteins by BiFC and FRET/FLIM in planta and with GFP/RFP traps in vitro. In the course of these studies we found that AtPII interacts in chloroplasts with itself as well as with known interactors like NAGK in dot-like aggregates, which we named PII foci. In these novel protein aggregates AtPII interacts also with yet unknown partners, which are known to be involved in plastidic protein degradation. Further studies revealed that the C-terminal part of AtPII is crucial for the formation of PII foci. Altogether, the presented results indicate a novel mode of interaction for PII proteins with other proteins in plants, which may be a new starting point for the elucidation of physiological functions of PII proteins in plants.

Acyclic olygoheteroaryl-based compounds represent a valuable class of ligands for nucleic acid recognition. In this regard, acyclic pyridyl polyoxazoles and polyoxadiazoles were recently identified as selective G-quadruplex stabilizing compounds with high cytotoxicity and promising anticancer activity. Herein, we describe the synthesis of a new family of polyheteroaryl oxadiazole/pyridine-ligands targeting DNA G-quadruplexes. In order to perform a structure-activity analysis identifying determinants of activity and selectivity, we followed a convergent synthetic pathway to modulate the nature and number of the heterocycles (1,3-oxazole vs 1,2,4-oxadiazole and pyridine vs benzene). Each ligand was evaluated towards secondary nucleic acid structures, which have been chosen as a prototype to mimic cancer-associated G-quadruplex structures (e.g., the human telomeric sequence, c-myc and c-kit promoters). Interesting, heptapyridyl-oxadiazole compounds showed preferential binding towards the telomeric sequence (22AG) in competitive conditions vs duplex DNA. In addition, G4-FID assays suggest a different binding mode from the classical stacking on the external G-quartet. Additionally, CD titrations in the presence of the two most promising compounds for affinity, TOxAzaPy and TOxAzaPhen, display a structural transition of 22AG in K-rich buffer. This investigation suggests that the pyridyl-oxadiazole motif is a promising recognition element for G-quadruplexes, combining seven heteroaryls in a single binding unit.

Pre-mRNA splicing is an essential process orchestrated by the spliceosome, a dynamic complex assembled stepwise on pre-mRNA. We have previously identified that USH1G protein SANS regulates pre-mRNA splicing by mediating the intra-nuclear transfer of the spliceosomal U4/U6.U5 tri-snRNP complex. During this process, SANS interacts with the U4/U6 and U5 snRNP-specific proteins PRPF31 and PRPF6 and regulates splicing, which is disturbed by variants of USH1G/SANS causative for human Usher syndrome (USH) the most common form of hereditary deaf-blindness.Here, we aimed to gain further insight into the molecular interaction of the splicing molecules PRPF31 and PRPF6 to the CENTn domain of SANS using fluorescence resonance energy transfer assays in cells and in silico deep learning-based protein structure predictions. This demonstrates that SANS directly binds via two distinct conserved regions of its CENTn to the two PRPFs. In addition, we provide evidence that these interactions occur sequentially and a conformational change of an intrinsically disordered region to a short α-helix of SANS CENTn2 is triggered by binding of PRPF6. Furthermore, we found that pathogenic variants of USH1G/SANS perturb the binding of SANS to both PRPFs implying a significance for the USH1G pathophysiology.

Treatment of various diseases, in particular cancer, usually needs targeting biologically active molecules at a selected subcellular compartment. We modified our previously developed modular nanotransporters (MNTs) for targeting mitochondria. The new MNTs are capable of binding to the protein predominantly localized on the outer mitochondrial membrane, Keap1. These MNTs possessing antiKeap1 monobody co-localize with mitochondria upon addition to the cells. They efficiently interact with Keap1 both in solution and within living cells. A conjugate of the MNT with a photosensitizer, chlorin e6, demonstrates significantly higher photocytotoxicity than chlorine e6 alone. We assume that MNTs of this kind can improve efficiency of therapeutic photosensitizers and radionuclides emitting short-range particles.

Most methods for measuring environmental lead (Pb) content are time consuming, expensive, hazardous, and restricted to specific analytical systems. To provide a facile, safe tool to detect Pb, we created pMet-lead, a portable fluorescence resonance energy transfer (FRET)-based Pb biosensor. pMet-lead comprises a 3D-printed frame housing a 405-nm laser diode — an excitation source for fluorescence emission images (YFP and CFP) — accompanied by optical filters, a customized sample holder with a Met-lead 1.44 M1 (the most recent version)-embedded biochip, and an optical lens aligned for smartphone compatibility. Measuring the emission ratios (Y/C) of the FRET component enables Pb detection with a dynamic range of nearly 2 (1.96), pMet-lead/Pb dissociation constant (K_d) 45.62 nM, and limit of detection 40 nM (0.832 μg/dL, 8.32 ppb). To mitigate earlier problems with lack of selectivity for Pb vs. zinc, we preincubated samples with tricine, a low-affinity zinc chelator. We validated pMet-lead measurements of characterized laboratory samples and unknown samples from six regions in Taiwan by inductively coupled plasma mass spectrometry (ICP-MS). Notably, two unknowns had Y/C ratios significantly higher than that of the control (3.48 ± 0.08 and 3.74 ± 0.12 vs. 2.79 ± 0.02), along with Pb concentrations (10.6 ppb and 15.24 ppb) above the WHO-permitted level of 10 ppb in tap water, while the rest four unknowns showing no detectable Pb upon ICP-MS. These results demonstrate that pMet-lead provides a rapid, sensitive means for on-site Pb detection in water from the environment and in living/drinking supply systems to prevent potential Pb poisoning.

Labelling techniques such as electron paramagnetic resonance spectroscopy and single-molecule fluorescence resonance energy transfer, allow access to distances in the range of tens of angstroms, corresponding to the size of proteins and small to medium-sized protein complexes. Such measurements do not require long-range ordering and are therefore applicable to systems with partial disorder. Data from spin-label-based measurements can be processed into distance distributions that provide information about the extent of such disorder. Using such information in modelling presents several challenges, including a small number of restraints, the influence of the label itself on the measured distance and distribution width, and balancing the fitting quality of the long-range restraints with the fitting quality of other restraint subsets. Starting with general considerations about integrative and hybrid structural modelling, this review provides an overview of recent approaches to these problems and identifies where further progress is needed.

C-terminal Src kinase (CSK) is the major inhibitory kinase for Src family kinases (SFKs) through phosphorylation of their C-tail tyrosine site, and regulates various cellular activities in association with SFKs functions. As a cytoplasmic protein, CSK need be recruited to the plasma membrane to regulate SFKs activities. The regulatory mechanism for CSK activity and its subcellular localization remains largely unclear. In this work, we developed a genetically encoded biosensor based on fluorescence resonance energy transfer (FRET) to visualize CSK activity in live cells. The biosensor with optimized substrate peptide confirmed crucial Arg107 site in CSK SH2 domain, and displayed sensitivity and specificity to CSK activity while little responses to co-transfected Src and Fyn. FRET measurements showed CSK having relatively mild level of kinase activity in comparison to Src and Fyn ones in rat airway smooth muscle cells. The biosensor tagged with different submembrane-targeting signals detected CSK activity at both non-lipid raft and lipid raft microregions, while showed higher FRET level at non-lipid ones. Co-transfected receptor-type protein tyrosine phosphatase alpha (PTPα) had inhibitory effect on the CSK FRET response. The biosensor didn’t detect obvious changes of CSK activity between metastatic cancer cells and normal ones. In conclusion, a novel FRET biosensor was generated to monitor CSK activity, and demonstrated CSK activity existing in both non-lipid and lipid raft membrane microregions while more present at non-lipid ones.

The homeostasis of cellular calcium is fundamental for many physiological activities, while calcium levels maintain inhomogeneous within cells. During the onset of asthma, epithelial and inflammatory cells secrete platelet derived growth factor (PDGF), inducing proliferation and migration of airway smooth muscle (ASM) to the epidermal layer, narrowing the airway. The regulation of ASM cells by PDGF is closely related to the conduction of calcium signals. In this work, we generated subcellular-targeted FRET biosensors to investigate calcium regulations at different compartments of ASM cells. PDGF-induced cytoplasmic calcium [Ca2+]C increase was attributed from both extracellular calcium influx and endoplasmic reticulum (ER) calcium [Ca2+]ER release, which was partially regulated by PLC-IP3R pathway. Interestingly, removal of extracellular calcium influx led to inhibited ER calcium release, likely through inhibitory effect on calcium-dependent activation of ER ryanodine receptor. Inhibition of L-type calcium channel on plasma membrane, or SERCA pump on ER resulted in both reduced [Ca2+]C and [Ca2+]ER from PDGF stimulation, while IP3R channel inhibition led to reduced [Ca2+]C only. Inhibited SERCA pump caused immediate [Ca2+]C increase and [Ca2+]ER decrease, indicating an active calcium exchange between cytosol and ER storage in resting cells. PDGF-induced calcium at outer mitochondrial membrane sub-region showed similar regulatory response to cytosolic calcium, not influenced by inhibition of mitochondrial calcium uniporter channel. Therefore, our work identified calcium flow pathways among extracellular medium, cell cytosol, and ER by regulatory calcium channels. Particularly, extracellular calcium flow has an essential function to fully activate ER calcium release.