Fluorescence-based Binding Assay Research Articles

Dually Labeled Neurotensin NTS1R Ligands for Probing Radiochemical and Fluorescence-Based Binding Assays.

The determination of ligand-receptor binding affinities plays a key role in the development process of pharmaceuticals. While the classical radiochemical binding assay uses radioligands, fluorescence-based binding assays require fluorescent probes. Usually, radio- and fluorescence-labeled ligands are dissimilar in terms of structure and bioactivity, and can be used in either radiochemical or fluorescence-based assays. Aiming for a close comparison of both assay types, we synthesized tritiated fluorescent neurotensin receptor ligands ([3H]13, [3H]18) and their nontritiated analogues (13, 18). The labeled probes were studied in radiochemical and fluorescence-based (high-content imaging, flow cytometry, fluorescence anisotropy) binding assays. Equilibrium saturation binding yielded well-comparable ligand-receptor affinities, indicating that all these setups can be used for the screening of new drugs. In contrast, discrepancies were found in the kinetic behavior of the probes, which can be attributed to technical differences of the methods and require further studies with respect to the elucidation of the underlying mechanisms.

Pulsatillic acid as a potential inhibitor of protein kinase C-alpha in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is a global health concern with a significant impact on morbidity and mortality. Small molecule inhibitors targeting genetic mutations like EGFR and ALK have shown promise in NSCLC treatment. This study focuses on Protein Kinase C-alpha (PKCα), implicated in NSCLC pathogenesis. Overexpression of PKCα correlates with advanced disease stages. Preclinical studies suggest its inhibition can suppress NSCLC cell growth. The research employs molecular docking to identify Pulsatillic acid (PA) as a potential PKCα inhibitor. ADMET predictions support PA's candidacy and PASS analysis and Swiss Target Prediction reveal its biological properties. Fluorescence-based binding assays demonstrate PA's inhibitory potency on PKCα, aligning with molecular docking findings. Cytotoxicity assays show PA's minimal impact on HEK-293 cell viability, with an IC50 of 21.03 μM in A549 cells. mRNA expression analysis in A549 cells indicates PA's potential inhibitory effect on PKCα. In conclusion, this study highlights that PA may emerge as a promising therapeutic candidate for NSCLC, emphasizing the need for further research, validation, and exploration of its translational potential. The study contributes valuable insights into NSCLC treatment strategies, emphasizing the significance of targeting PKCα.

Phosphorylation by Protein Kinase C Weakens DNA-Binding Affinity and Folding Stability of the HMGB1 Protein.

The HMGB1 protein typically serves as a DNA chaperone that assists DNA-repair enzymes and transcription factors but can translocate from the nucleus to the cytoplasm or even to extracellular space upon some cellular stimuli. One of the factors that triggers the translocation of HMGB1 is its phosphorylation near a nuclear localization sequence by protein kinase C (PKC), although the exact modification sites on HMGB1 remain ambiguous. In this study, using spectroscopic methods, we investigated the HMGB1 phosphorylation and its impact on the molecular properties of the HMGB1 protein. Our nuclear magnetic resonance (NMR) data on the full-length HMGB1 protein showed that PKC specifically phosphorylates the A-box domain, one of the DNA binding domains of HMGB1. Phosphorylation of S46 and S53 was particularly efficient. Over a longer reaction time, PKC phosphorylated some additional residues within the HMGB1 A-box domain. Our fluorescence-based binding assays showed that the phosphorylation significantly reduces the binding affinity of HMGB1 for DNA. Based on the crystal structures of HMGB1-DNA complexes, this effect can be ascribed to electrostatic repulsion between the negatively charged phosphate groups at the S46 side chain and DNA backbone. Our data also showed that the phosphorylation destabilizes the folding of the A-box domain. Thus, phosphorylation by PKC weakens the DNA-binding affinity and folding stability of HMGB1.

The Female-Biased General Odorant Binding Protein 2 of Semiothisa cinerearia Displays Binding Affinity for Biologically Active Host Plant Volatiles.

Herbivorous insects rely on volatile chemical cues from host plants to locate food sources and oviposition sites. General odorant binding proteins (GOBPs) are believed to be involved in the detection of host plant volatiles. In the present study, one GOBP gene, ScinGOBP2, was cloned from the antennae of adult Semiothisa cinerearia. Reverse-transcription PCR and real-time quantitative PCR analysis revealed that the expression of ScinGOBP2 was strongly biased towards the female antennae. Fluorescence-based competitive binding assays revealed that 8 of the 27 host plant volatiles, including geranyl acetone, decanal, cis-3-hexenyl n-valerate, cis-3-hexenyl butyrate, 1-nonene, dipentene, α-pinene and β-pinene, bound to ScinGOBP2 (KD = 2.21-14.94 μM). The electrical activities of all eight ScinGOBP2 ligands were confirmed using electroantennography. Furthermore, oviposition preference experiments showed that eight host volatiles, such as decanal, cis-3-hexenyl n-valerate, cis-3-hexenyl butyrate, and α-pinene, had an attractive effect on female S. cinerearia, whereas geranyl acetone, 1-nonene, β-pinene, and dipentene inhibited oviposition in females. Consequently, it can be postulated that ScinGOBP2 may be implicated in the perception of host plant volatiles and that ScinGOBP2 ligands represent significant semiochemicals mediating the interactions between plants and S. cinerearia. This insight could facilitate the development of a chemical ecology-based approach for the management of S. cinerearia.

Identification of small molecule inhibitors of penicillin-binding protein 2a of methicillin-resistant Staphylococcus aureus for the therapeutics of bacterial infection.

The penicillin binding protein 2a (PBP2a) is a key enzyme associated with bacterial cell wall synthesis and bacterial infection. Therefore, targeting PBPa2 offers a promising approach for the therapeutics of bacterial resistance and infection. This study presents a comprehensive analysis of alpha-mangostin as a potential inhibitor of PBPa2. Molecular docking simulations revealed a strong binding affinity between alpha-mangostin and PBP2a, with an affinity score of -6.01 kcal/mol. Notably, alpha-mangostin formed a preferential hydrogen bond with THR216 of PBP2a, alongside several other polar and hydrophobic interactions. ADME and Toxicity predictions indicated that alpha-mangostin possesses favourable pharmacokinetic properties, suggesting its potential as a therapeutic agent. PASS analysis further highlighted its broad range of favourable biological properties. SwissTargetPrediction analysis reinforced these findings, indicating alpha-mangostin's association with various biological processes. Cell toxicity assays demonstrated that alpha-mangostin had no significant impact on the viability of HEK-293 cells, suggesting its potential safety for further development. The IC50 value for alpha-mangostin was found to be 33.43µM. Fluorescence-based binding assays showed that alpha-mangostin effectively inhibited PBP2a activity in a concentration-dependent manner, supporting its role as an inhibitor. In conclusion, the results suggest alpha-mangostin as a promising candidate for inhibiting PBP2a. Further, extensive studies are warranted to explore its clinical applications.

Host-functionalization of macrin nanoparticles to enable drug loading and control tumor-associated macrophage phenotype.

Macrophages are critical regulators of the tumor microenvironment and often present an immuno-suppressive phenotype, supporting tumor growth and immune evasion. Promoting a robust pro-inflammatory macrophage phenotype has emerged as a therapeutic modality that supports tumor clearance, including through synergy with immune checkpoint therapies. Polyglucose nanoparticles (macrins), which possess high macrophage affinity, are useful vehicles for delivering drugs to macrophages, potentially altering their phenotype. Here, we examine the potential of functionalized macrins, synthesized by crosslinking carboxymethyl dextran with L-lysine, as effective carriers of immuno-stimulatory drugs to tumor-associated macrophages (TAMs). Azide groups incorporated during particle synthesis provided a handle for click-coupling of propargyl-modified β-cyclodextrin to macrins under mild conditions. Fluorescence-based competitive binding assays revealed the ability of β-cyclodextrin to non-covalently bind to hydrophobic immuno-stimulatory drug candidates (Keq ~ 103 M-1), enabling drug loading within nanoparticles. Furthermore, transcriptional profiles of macrophages indicated robust pro-inflammatory reprogramming (elevated Nos2 and Il12; suppressed Arg1 and Mrc1 expression levels) for a subset of these immuno-stimulatory agents (UNC2025 and R848). Loading of R848 into the modified macrins improved the drug's effect on primary murine macrophages by three-fold in vitro. Intravital microscopy in IL-12-eYFP reporter mice (24h post-injection) revealed a two-fold enhancement in mean YFP fluorescence intensity in macrophages targeted with R848-loaded macrins, relative to vehicle controls, validating the desired pro-inflammatory reprogramming of TAMs in vivo by cell-targeted drug delivery. Finally, in an intradermal MC38 tumor model, cyclodextrin-modified macrin NPs loaded with immunostimulatory drugs significantly reduced tumor growth. Therefore, efficient and effective repolarization of tumor-associated macrophages to an M1-like phenotype-via drug-loaded macrins-inhibits tumor growth and may be useful as an adjuvant to existing immune checkpoint therapies.

A fluorescence-based binding assay for proteins using the cell surface as a sensing platform.

Protein-protein interaction (PPI) analysis is very important for elucidating the functions of proteins because many proteins execute their functions in living cells by interacting with one another. In PPI analysis, methods using the sensor chips are widely employed to obtain quantitative data. However, these methods require that the target proteins be immobilized on the sensor chips, and the immobilization processes can affect the binding of the target proteins to their binding partners. In the present work, we propose a PPI analysis system in which the surface of the living cells is utilized as a sensing platform. In our approach, the target protein is displayed on the cell surface by expressing it as a fusion protein with a membrane protein, and the PPI analysis is then conducted by applying its binding partner labeled with a fluorescent dye to the cell surface. We have constructed a model of this binding analysis system using the interaction between biotin protein ligase (BPL) and biotin carboxyl carrier protein (BCCP), where BCCP was displayed on the cell surface and BPL labeled with fluorescein was applied to the cell surface. Here, a red fluorescent protein, mApple, was attached to the C-terminus of the fusion protein of BCCP with a membrane protein. We evaluated the binding level of the labeled BPL by using the intensity ratios of fluorescence from fluorescein to that from mApple. We found that the binding level of the labeled BPL was stably evaluated at least across 60min observation period and estimated the binding dissociation constant between BPL and BCCP by equilibrium analysis to be 0.33 ± 0.05μM.

Evidence of DISC1 as an arsenic binding protein and implications regarding its role as a translational activator.

Disrupted-in-schizophrenia-1 (DISC1) is a scaffolding protein that plays a pivotal role in orchestrating signaling pathways involved in neurodevelopment, neural migration, and synaptogenesis. Among those, it has recently been reported that the role of DISC1 in the Akt/mTOR pathway can shift from a global translational repressor to a translational activator in response to oxidative stress induced by arsenic. In this study we provide evidence that DISC1 can directly bind arsenic via a C-terminal cysteine motif (C-X-C-X-C). A series of fluorescence-based binding assays were conducted with a truncated C-terminal domain construct of DISC1 and a series of single, double, and triple cysteine mutants. We found that arsenous acid, a trivalent arsenic derivative, specifically binds to the C-terminal cysteine motif of DISC1 with low micromolar affinity. All three cysteines of the motif are required for high-affinity binding. Electron microscopy experiments combined with in silico structural predictions reveal that the C-terminal of DISC1 forms an elongated tetrameric complex. The cysteine motif is consistently predicted to be located within a loop, fully exposed to solvent, providing a simple molecular framework to explain the high-affinity of DISC1 toward arsenous acid. This study sheds light on a novel functional facet of DISC1 as an arsenic binding protein and highlights its potential role as both a sensor and translational modulator within Akt/mTOR pathway.

Expression and functional analysis of an odorant binding protein PopeOBP16 from Phthorimaea operculella (Zeller)

Odorant binding proteins (OBPs) are essential proteins in the peripheral olfactory system, responsible for odorant recognition and transport to olfactory receptors. Phthorimaea operculella (potato tuber moth) is an important oligophagous pest on Solanaceae crops in many countries and regions. PopeOBP16 is one of the OBPs in potato tuber moth. This study examined the expression profiles of PopeOBP16. The results of qPCR indicated that PopeOBP16 was highly expressed in the antennae of adults, especially in males, suggesting that it may be involved in odor recognition in adults. The electroantennogram (EAG) was used to screen candidate compounds with the antennae of P. operculella. The relative affinities of PopeOBP16 to 27 host volatiles and two sex pheromone components with the highest relative EAG responses were examined with competitive fluorescence-based binding assays. PopeOBP16 had the strongest binding affinity with the plant volatiles: nerol, 2-phenylethanol, linalool, 1,8-cineole, benzaldehyde, β-pinene, d-limonene, terpinolene, α-terpinene, and the sex pheromone component trans-4, cis-7, cis-10-tridecatrien-1-ol acetate. The results provide a foundation for further research into the functioning of the olfactory system and the potential development of green chemistry for control of the potato tuber moth.

Small-Molecule Fluorescent Ligands for the CXCR4 Chemokine Receptor.

The C-X-C chemokine receptor type 4, or CXCR4, is a chemokine receptor found to promote cancer progression and metastasis of various cancer cell types. To investigate the pharmacology of this receptor, and to further elucidate its role in cancer, novel chemical tools are a necessity. In the present study, using classic medicinal chemistry approaches, small-molecule-based fluorescent probes were designed and synthesized based on previously reported small-molecule antagonists. Here, we report the development of three distinct chemical classes of fluorescent probes that show specific binding to the CXCR4 receptor in a novel fluorescence-based NanoBRET binding assay (pKD ranging 6.6-7.1). Due to their retained affinity at CXCR4, we furthermore report their use in competition binding experiments and confocal microscopy to investigate the pharmacology and cellular distribution of this receptor.

Inhibition effect of 1-acetoxy-6α-(2-methylbutyryl)eriolanolide toward soluble epoxide hydrolase: Multispectral analysis, molecular dynamics simulation, biochemical, and in vitro cell-based studies

Soluble epoxide hydrolase (sEH) serves as a potential target in inflammation-related diseases. Based on the bioactivity-guided separation, a new sesquiterpenoid inulajaponoid A (1) was isolated from Inula japonica with a sEH inhibitory effect, together with five known compounds, such as 1-O-acetyl-6-O-isobutyrylbritannilactone (2), 6β-hydroxytomentosin (3), 1β,8β-dihydroxyeudesma-4(15),11(13)-dien-12,6α-olide (4), (4S,6S,7S,8R)-1-O-acetyl-6-O-(3-methylvaleryloxy)-britannilactone (5), and 1-acetoxy-6α-(2-methylbutyryl)eriolanolide (6). Among them, compounds 1 and 6 were assigned as mixed and uncompetitive inhibitors, respectively. The result of immunoprecipitation (IP)-MS demonstrated the specific binding of compound 6 to sEH in the complex system, which was further confirmed by the fluorescence-based binding assay showing its equilibrium dissociation constant (Kd = 2.43 μM). The detail molecular stimulation revealed the mechanism of action of compound 6 with sEH through the hydrogen bond of amino acid residue Gln384. Furthermore, this natural sEH inhibitor (6) could suppress the MAPK/NF-κB activation to regulate inflammatory mediators, such as NO, TNF-α, and IL-6, which confirmed the anti-inflammatory effect of inhibition of sEH by 6. These findings provided a useful insight to develop sEH inhibitors upon the sesquiterpenoids.

4-Aminoquinolines modulate RNA structure and function: Pharmacophore implications of a conformationally restricted polyamine

RNA structure plays an important role in regulating cellular function and there is a significant emerging interest in targeting RNA for drug discovery. Here we report the identification of 4-aminoquinolines as modulators of RNA structure and function. Aminoquinolines have a broad range of pharmacological activities, but their specific mechanism of action is often not fully understood. Using electrophoretic mobility shift assays and enzymatic probing we identified 4-aminoquinolines that bind the stem-loop II motif (s2m) of SARS-CoV-2 RNA site-specifically and induce dimerization. Using fluorescence-based RNA binding and T-box riboswitch functional assays we identified that hydroxychloroquine binds the T-box riboswitch antiterminator RNA element and inhibits riboswitch function. Based on its structure and riboswitch dose-response activity we identified that the antagonist activity of hydroxychloroquine is consistent with it being a conformationally restricted analog of the polyamine spermidine. Given the known role that polyamines play in RNA function, the identification of an RNA binding ligand with the pharmacophore of a conformationally restricted polyamine has significant implications for further elucidation of RNA structure-function relationships and RNA-targeted drug discovery.

Fragment Ligands of the m6A-RNA Reader YTHDF2.

We report 17 small-molecule ligands that compete with N6-methyladenosine (m6A) for binding to the m6A-reader domain of YTHDF2 (YT521-B homology domain family 2). We determined their binding mode at high resolution by X-ray crystallography and quantified their affinity by a fluorescence-based binding assay. 6-Cyclopropyluracil and a pyrazolopyrimidine derivative have favorable ligand efficiencies of 0.47 and 0.38 kcal mol–1 per non-hydrogen atom, respectively. They represent useful starting points for hit optimization.

Design and Development of Benzothiazole-Based Fluorescent Probes for Selective Detection of Aβ Aggregates in Alzheimer's Disease.

The formation and accumulation of amyloid beta (Aβ) peptide are considered the crucial events that are responsible for the progression of Alzheimer's disease (AD). Herein, we have designed and synthesized a series of fluorescent probes by using electron acceptor-donor end groups interacting with a π-conjugating system for the detection of Aβ aggregates. The chemical structure of these probes denoted as RMs, having a conjugated π-system (C═C), showed a maximum emission in PBS (>600 nm), which is the best range for a fluorescent imaging probe. Among all these probes, RM-28 showed an excellent fluorescence property with an emission maximum of >598 nm upon binding to Aβ aggregates. RM-28 also showed high sensitivity (7.5-fold) and high affinities toward Aβ aggregates (Kd = 175.69 ± 4.8 nM; Ka = 0.5 × 107 M-1). It can cross the blood-brain barrier of mice efficiently. The affinity of RM-28 toward Aβ aggregates was observed in 3xTg-AD brain sections of the hippocampus and cortex region using a fluorescent imaging technique, as well as an in vitro fluorescence-based binding assay with Aβ aggregates. Moreover, RM-28 is highly specific to Aβ aggregates and does not bind with intracellular proteins like bovine serum albumin (BSA) and α-synuclein (α-Syn) aggregates. The results indicate that the probe RM-28 emerges as an efficient and veritable highly specific fluorescent probe for the detection of Aβ aggregates in both in vitro and in vivo model systems.

Abstract 5639: Computational prediction of MHC anchor locations guide neoantigen prediction and prioritization

Abstract Neoantigens are novel peptide sequences resulting from somatic mutations in tumors that upon loading onto major histocompatibility complex (MHC) molecules allow recognition by T cells. Accurate neoantigen identification is thus critical for designing cancer vaccines and predicting response to immunotherapies. Neoantigen identification and prioritization relies on correctly inferring whether the presenting peptide sequence can successfully induce an immune response. As the majority of somatic mutations are SNVs, changes between wildtype and mutant peptide are subtle and require cautious interpretation. An important, yet underappreciated, variable in neoantigen-prediction pipelines is the mutation position within the peptide relative to its anchor positions for the patient’s specific HLA alleles. While a subset of peptide positions are presented to the T-cell receptor for recognition, others are responsible for anchoring to the MHC, making these positional considerations critical for predicting T-cell responses. However, a systematic method for determining anchor locations for the wide range of HLA alleles present in the population and application of these to evaluate MT/WT peptide pairs arising in tumors has not been reported. As a result, many neoantigen studies have either failed to adequately consider this crucial factor or have used conventional assumptions to guide their neoantigen identification process. Here, we provide a computational workflow for predicting anchor locations for a wide range of HLA alleles, using a reference dataset generated from clinical and The Cancer Genome Atlas (TCGA) patient samples. We calculated high probability anchor positions for different peptide lengths for over 300 common HLA alleles. Analysis of these results showed clusters of different anchor trends among the HLA alleles analyzed. A subset of these HLA anchor results were orthogonally validated using protein crystallography structures. Analysis of 923 tumor samples showed that 7-41% of neoantigen candidates were potentially misclassified in the neoantigen selection process and can be rescued using allele-specific knowledge of anchor positions. These anchor predictions are currently undergoing experimental validation using both peptide-MHC stability assays as well as fluorescence-based competition binding assays. By incorporating our anchor prediction results into neoantigen prediction pipelines, such as pVACtools, we hope to formalize and streamline the identification process for relevant clinical studies. Citation Format: Huiming Xia, Joshua McMichael, Michelle Becker-Hapak, Onyinyechi C. Onyeador, Rico Buchli, Ethan McClain, Patrick Pence, Suangson Supabphol, Megan M. Richters, Anamika Basu, Cody A. Ramirez, Cristina Puig-Saus, Kelsy C. Cotto, Jasreet Hundal, Susanna Kiwala, S. Peter Goedegebuure, Tanner M. Johanns, Gavin P. Dunn, Antoni Ribas, Christopher A. Miller, William Gillanders, Todd A. Fehniger, Obi L. Griffith, Malachi Griffith. Computational prediction of MHC anchor locations guide neoantigen prediction and prioritization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5639.

Mechanistic Insight into Binding of Huperzine A with Human Serum Albumin: Computational and Spectroscopic Approaches.

Human serum albumin (HSA) is the most abundant protein in plasma synthesized by the liver and the main modulator of fluid distribution between body compartments. It has an amazing capacity to bind with multiple ligands, offering a store and transporter for various endogenous and exogenous compounds. Huperzine A (HpzA) is a natural sesquiterpene alkaloid found in Huperzia serrata and used in various neurological conditions, including Alzheimer’s disease (AD). This study elucidated the binding of HpzA with HSA using advanced computational approaches such as molecular docking and molecular dynamic (MD) simulation followed by fluorescence-based binding assays. The molecular docking result showed plausible interaction between HpzA and HSA. The MD simulation and principal component analysis (PCA) results supported the stable interactions of the protein–ligand complex. The fluorescence assay further validated the in silico study, revealing significant binding affinity between HpzA and HSA. This study advocated that HpzA acts as a latent HSA binding partner, which may be investigated further in AD therapy in experimental settings.

Odorant-Binding Protein 1 Plays a Crucial Role in the Olfactory Response of Bemisia tabaci to R-Curcumene.

The cultivated tomato Solanum lycopersicum suffered a severe attack by the whitefly Bemisia tabaci (Gennadius), causing damage to leaves by feeding as well as transmitting the tomato yellow leaf curl virus (TYLCV), while the wild tomato S. habrochaites is considerably less appealing to this insect species. It is reported that B. tabaci shows innate avoidance to R-curcumene, which is produced naturally by S. habrochaites. However, the mechanisms involved in the avoidance behavior of B. tabaci in response to this chiral compound are still unclear yet. In this study, the functional and binding characterization of odorant-binding protein 1 of B. tabaci (BtOBP1) were examined in vivo and in vitro against R-curcumene. The obtained results showed that BtOBP1 exhibits specific binding activity to R-curcumene, which acts as repellents to B. tabaci. By using a fluorescence-based binding assay, the difference of binding-affinity for R-curcumene between wild type BtOBP1 and the mutant BtOBP1 to R-curcumene was performed, which resulted in a single amino acid mutation (ASN108 > SER); moreover, BtOBP1-N108 displays significantly decreased binding affinities to R-curcumene. Most interestingly, a knock-down experiment with the BtOBP1 showed that the whitefly responses to R-curcumene are impaired. This study illustrated that BtOBP1 is a crucial protein involved in the perception and discrimination of R-curcumene. Our findings may provide an excellent chance of finding a suitable antagonist of eco-friendly features that can block the perception of chemosensory signals in insects, preventing behaviors like food-finding.

High-Throughput Selection and Characterisation of Aptamers on Optical Next-Generation Sequencers.

Aptamers feature a number of advantages, compared to antibodies. However, their application has been limited so far, mainly because of the complex selection process. ‘High-throughput sequencing fluorescent ligand interaction profiling’ (HiTS–FLIP) significantly increases the selection efficiency and is consequently a very powerful and versatile technology for the selection of high-performance aptamers. It is the first experiment to allow the direct and quantitative measurement of the affinity and specificity of millions of aptamers simultaneously by harnessing the potential of optical next-generation sequencing platforms to perform fluorescence-based binding assays on the clusters displayed on the flow cells and determining their sequence and position in regular high-throughput sequencing. Many variants of the experiment have been developed that allow automation and in situ conversion of DNA clusters into base-modified DNA, RNA, peptides, and even proteins. In addition, the information from mutational assays, performed with HiTS–FLIP, provides deep insights into the relationship between the sequence, structure, and function of aptamers. This enables a detailed understanding of the sequence-specific rules that determine affinity, and thus, supports the evolution of aptamers. Current variants of the HiTS–FLIP experiment and its application in the field of aptamer selection, characterisation, and optimisation are presented in this review.

Cardiolipin in the spotlight: Quantitative analysis and fluorescence-based competitive binding assay

Cardiolipin (CL) is a key phospholipid responsible for mitochondrial function and cristae integrity. The CL level is associated with various diseases characterized by mitochondrial dysfunction, including ischemic heart diseases and cancer. CL is an attractive target for mitochondria-specific drugs, but unnecessary interaction with CL might lead to detrimental side effects such as heart failure and kidney dysfunction. Thus, a simple and robust method for CL quantification and a reliable assay for the determination of drug affinity for CL are desired. We report a new fluorescent CL-specific probe with impressive photophysical properties that allows CL quantification in mitochondrial fractions isolated from cell and tissue homogenates and enables estimates of drug affinity for CL in the first fluorescence-based competitive binding assay. It was found that CL concentration is elevated in mitochondrial fractions isolated from cancer cells and cells with high proliferation rate (up to 108.5 ± 16.0 nmol/mg prot in mouse colon carcinoma cells, CT-26). CL concentration in mitochondria from brain tissue (66.11 ± 5.78 nmol/mg prot) is circa twice higher than in heart and kidney mitochondria (37.49 ± 8.69 and 33.95 ± 5.32 nmol/mg prot, respectively.) Generally, positively charged substances bind with CL, but their affinity is highly variable with EC50 values ranging from sub-micromolar to millimolar concentration.

Identification and functional characterization of odorant-binding proteins 69a and 76a of Drosophila suzukii

The fruit fly Drosophila suzukii is a fruit crop pest that causes a severe economic threat to soft summer fruit worldwide. The male sex pheromone, cis-vaccenyl acetate (cVA) has multiple functions in intra-species communication in Drosophila melanogaster, which is required in male to suppress male-male courtship. D. suzukii males do not produce cVA; however, the odorant receptor for cVA (Or67d) is still functional. The lack of cVA in D. suzukii casts the question of whether this pheromone might have been replaced by another compound similar to cVA that disrupts mating in D. suzukii. In order to address this question, we cloned two D. suzukii adult antenna-specific odorant-binding proteins (OBPs) DsOBP69a and DsOBP76a and aligned with their D. melanogaster orthologues. Moreover, we examined the binding properties of the newly identified recombinant proteins against 26 potential ligands including cVA, using the fluorescence-based ligand binding assay. The alignment showed that DsOBP69a and DsOBP76a, have six conserved cysteines and belong to the classic OBP family. Furthermore, our results revealed that cVA did not bind to DsOBP69a or DsOBP76a proteins. Interestingly, the floral odorant β-ionone and the bitter substance berberine chloride and coumarin displayed high binding ability. It is also worth noting that DsOBP69a and DsOBP76a have different affinities to (Z)-7-Tricosene that may reflect different functional roles. These findings suggest that DsOBP69a and DsOBP76a are potentially involved in olfaction and gustation of D. suzukii.