Fluorophore Labeling Research Articles

Nanobodies as novel tools to monitor the mitochondrial fission factor Drp1.

In cells, mitochondria undergo constant fusion and fission. An essential factor for fission is the mammalian dynamin-related protein 1 (Drp1). Dysregulation of Drp1 is associated with neurodegenerative diseases including Parkinson's, cardiovascular diseases and cancer, making Drp1 a pivotal biomarker for monitoring mitochondrial status and potential pathophysiological conditions. Here, we developed nanobodies (Nbs) as versatile binding molecules for proteomics, advanced microscopy and live cell imaging of Drp1. To specifically enrich endogenous Drp1 with interacting proteins for proteomics, we functionalized high-affinity Nbs into advanced capture matrices. Furthermore, we detected Drp1 by bivalent Nbs combined with site-directed fluorophore labelling in super-resolution STORM microscopy. For real-time imaging of Drp1, we intracellularly expressed fluorescently labelled Nbs, so-called chromobodies (Cbs). To improve the signal-to-noise ratio, we further converted Cbs into a "turnover-accelerated" format. With these imaging probes, we visualized the dynamics of endogenous Drp1 upon compound-induced mitochondrial fission in living cells. Considering the wide range of research applications, the presented Nb toolset will open up new possibilities for advanced functional studies of Drp1 in disease-relevant models.

Nitroreductase-Triggered Fluorophore Labeling of Cells and Tissues under Hypoxia.

Several reductases, including nitroreductase, are upregulated under hypoxic conditions characterized by an oxygen-deficient microenvironment. Given that hypoxia is a prominent feature of solid tumors, our investigation focused on developing a bioconjugative probe designed for staining tissue under hypoxic conditions, particularly activated by nitroreductase. This probe, developed using our trigger-release-bioconjugation system rooted in the ortho-quinone methide chemistry, exhibited selective activation by nitroreductase and fluorophore labeling within mitochondria and endoplasmic reticulum. As a result, it displayed sustained fluorescence that persisted even after washing steps in cells and tissues. We applied this innovative probe to stain mouse kidney tissue in an acute kidney injury model induced by inadequate oxygen supply. Among various organ tissues examined, only kidney tissue showed significantly higher fluorescence in the injury model compared with the control tissue, as revealed by two-photon microscopic imaging. This research presents a promising avenue for the development of practical staining agents for image-guided tumor surgery.

In Migratio Noncovalent Fluorophore Labeling of Proteins by Propidium Iodide in Sodium Dodecyl Sulfate Capillary Gel Electrophoresis.

Sodium dodecyl sulfate capillary gel electrophoresis is one of the frequently used methods for size-based protein separation in molecular biology laboratories and the biopharmaceutical industry. To increase throughput, quite a few multicapillary electrophoresis systems have been recently developed, but most of them only support fluorescence detection, requiring fluorophore labeling of the sample proteins. To avoid the time-consuming derivatization reaction, we developed an on-column labeling approach utilizing propidium iodide for the first time in SDS-CGE of proteins, a dye only used before for nucleic acid analysis. As a key ingredient of the gel-buffer system, the oppositely migrating positively charged propidium ligand in migratio complexes with the SDS-proteins, therefore, supports in situ labeling during the electrophoretic separation process, not requiring any extra pre- or postcolumn derivatization step. A theoretical treatment is given to shed light on the basic principles of this novel online labeling process, also addressing the influence of propidium iodide on the electroosmotic flow, resulting in reduced retardation. The concept of propidium labeling in SDS-CGE was first demonstrated using a commercially available protein sizing ladder ranging from 6.5 to 200 kDa with different isoelectric points and post-translational modifications. Considering the increasing number of protein therapeutics on the market next, we focused on the labeling optimization of a therapeutic monoclonal antibody and its subunits, including the addition of the nonglycosylated heavy chain. Peak efficiency and resolution were compared between noncovalent and covalent labeling. The effect of ligand concentration on the effective and apparent electrophoretic mobility, the resulting peak area, and the resolution were all evaluated in view of the theoretical considerations. The best detection sensitivity for the intact monoclonal antibody was obtained by using 200 μg/mL propidium iodide in the separation medium (LOD 2 μg/mL, 1.35 × 10-8 M) with excellent detection linearity over 3 orders of magnitude. On the other hand, the resolution between the biopharmaceutical protein test mixture components containing the intact and subunit fragments of the therapeutic monoclonal antibody was very good in the ligand concentration range of 50-200 μg/mL, but using the local maximum at 100 μg/mL for the nonglycosylated/glycosylated heavy chain pair is recommended. The figures of merit, including precision, sensitivity, detection linear range, and resolution for a sample mixture in hand, can be optimized by varying the propidium iodide concentration in the gel-buffer system, as demonstrated in this paper.

Potent Immunomodulators Developed from an Unstable Bacterial Metabolite of Vitamin B2 Biosynthesis

AbstractBacterial synthesis of vitamin B2 generates a by‐product, 5‐(2‐oxopropylideneamino)‐d‐ribityl‐aminouracil (5‐OP‐RU), with potent immunological properties in mammals, but it is rapidly degraded in water. This natural product covalently bonds to the key immunological protein MR1 in the endoplasmic reticulum of antigen presenting cells (APCs), enabling MR1 refolding and trafficking to the cell surface, where it interacts with T cell receptors (TCRs) on mucosal associated invariant T lymphocytes (MAIT cells), activating their immunological and antimicrobial properties. Here, we strategically modify this natural product to understand the molecular basis of its recognition by MR1. This culminated in the discovery of new water‐stable compounds with extremely powerful and distinctive immunological functions. We report their capacity to bind MR1 inside APCs, triggering its expression on the cell surface (EC50 17 nM), and their potent activation (EC50 56 pM) or inhibition (IC50 80 nM) of interacting MAIT cells. We further derivatize compounds with diazirine‐alkyne, biotin, or fluorophore (Cy5 or AF647) labels for detecting, monitoring, and studying cellular MR1. Computer modeling casts new light on the molecular mechanism of activation, revealing that potent activators are first captured in a tyrosine‐ and serine‐lined cleft in MR1 via specific pi‐interactions and H‐bonds, before more tightly attaching via a covalent bond to Lys43 in MR1. This chemical study advances our molecular understanding of how bacterial metabolites are captured by MR1, influence cell surface expression of MR1, interact with T cells to induce immunity, and offers novel clues for developing new vaccine adjuvants, immunotherapeutics, and anticancer drugs.

Potent Immunomodulators Developed from an Unstable Bacterial Metabolite of Vitamin B2 Biosynthesis.

Bacterial synthesis of vitamin B2 generates a by-product, 5-(2-oxopropylideneamino)-d-ribityl-aminouracil (5-OP-RU), with potent immunological properties in mammals, but it is rapidly degraded in water. This natural product covalently bonds to the key immunological protein MR1 in the endoplasmic reticulum of antigen presenting cells (APCs), enabling MR1 refolding and trafficking to the cell surface, where it interacts with T cell receptors (TCRs) on mucosal associated invariant T lymphocytes (MAIT cells), activating their immunological and antimicrobial properties. Here, we strategically modify this natural product to understand the molecular basis of its recognition by MR1. This culminated in the discovery of new water-stable compounds with extremely powerful and distinctive immunological functions. We report their capacity to bind MR1 inside APCs, triggering its expression on the cell surface (EC50 17 nM), and their potent activation (EC50 56 pM) or inhibition (IC50 80 nM) of interacting MAIT cells. We further derivatize compounds with diazirine-alkyne, biotin, or fluorophore (Cy5 or AF647) labels for detecting, monitoring, and studying cellular MR1. Computer modeling casts new light on the molecular mechanism of activation, revealing that potent activators are first captured in a tyrosine- and serine-lined cleft in MR1 via specific pi-interactions and H-bonds, before more tightly attaching via a covalent bond to Lys43 in MR1. This chemical study advances our molecular understanding of how bacterial metabolites are captured by MR1, influence cell surface expression of MR1, interact with T cells to induce immunity, and offers novel clues for developing new vaccine adjuvants, immunotherapeutics, and anticancer drugs.

A highly cost-effective and sensitive biosensing platform: Fuel-propelled DNA nanomachines modulate fluorescence switching of DNA silver nanoclusters

It is of great significance to develop a low-cost, simple but highly sensitive fluorescence sensing platform for low-abundance molecule detection. However, traditional organic fluorophore labeling often requires expensive and sophisticated synthesis, making it difficult to use in resource-limited areas. Besides, the traditional “one-to-one” detection mechanism presents an obstacle to sensitive detection of low-level molecules, because one target only triggers one signal. To tackle these predicaments, in this work, a brightly red-emissive three-stranded DNA stabilized silver nanocluster (Ag NC) has been successfully synthesized. However, the fluorescence emission of Ag NCs can be darkened when it is in a loose single-stranded DNA. Building on this finding, we elaborately engineer a highly cost-effective and sensitive biosensing platform, which exploits a target-triggered and fuel-driven DNA nanomachine to precisely manipulate the structural transformation of Ag NCs from the rigid three-stranded DNA structures to the flexible single-stranded DNA, accordingly achieving the fluorescence switching for amplified sensing of low-abundance molecules. It is worth noting that nucleases and complex secondary structures are not involved in the entire system, offering a simple and flexible design. Furthermore, the novel and red-emissive DNA Ag NCs are employed as signal reporting, avoiding expensive and time-consuming organic fluorophore labeling. Experiment results demonstrate that this newly developed biosensing platform has a high signal-to-noise (∼15-folds), low limit of detection (1.59 pM), and good specificity to H5N1 DNA. Importantly, it can be used for H5N1 DNA detection in complex biological samples with a satisfactory recovery.

Direct and accurate miRNA detection based on CRISPR/Cas13a-triggered exonuclease-iii-assisted colorimetric assay

The abnormally expressed microRNAs (miRNAs) serve as crucial indicators for disease diagnosis and are strongly associated with the progression of various diseases. The quantification of miRNAs is highly significant for the clinical diagnosis and treatment of various types of cancers. This study utilizes the CRISPR/Cas13a system, which combines CRISPR RNA (crRNA) and CRISPR-associated Cas13a, to recognize the miRNA directly and specifically, thus activating the trans-cleavage activity of Cas13a. By integrating the CRISPR-Cas13a system with an exonuclease-iii (Exo-iii)-assisted chain cleavage of silver ions (Ag+)-aptamer and an Ag+ -based color reaction, a method for the detection of miRNA that is specific and sensitive is developed. This approach demonstrated exceptional sensitivity in detecting miRNA, with a low detection limit of 5.12 fM. Moreover, the suggested method was effectively utilized to precisely measure the amount of miRNA in intricate biological samples, demonstrating its practical applicability. Furthermore, due to its elevated sensitivity and simple probe design and fluorophore labeling, the suggested colorimetric technique holds great potential for use in clinical diagnostics.

Differential scanning fluorimetry to assess PFAS binding to bovine serum albumin protein.

The rapid screening of protein binding affinity for poly- and perfluoroalkyl substances (PFAS) benefits risk assessment and fate and transport modelling. PFAS are known to bioaccumulate in livestock through contaminated food and water. One excretion pathway is through milk, which may be facilitated by binding to milk proteins such as bovine serum albumin (BSA). We report a label-free differential scanning fluorimetry approach to determine PFAS-BSA binding over a broad temperature range. This method utilizes the tryptophan residue within the protein binding pocket as an intrinsic fluorophore, eliminating the need for fluorophore labels that may influence binding. BSA association constants were determined by (a) an equilibrium-based model at the melting temperature of BSA and (b) the Hill adsorption model to account for temperature dependent binding and binding cooperativity. Differences in binding between PFAS and fatty acid analogs revealed that a combination of size and hydrophobicity drives PFAS binding.

Activity-Based Protein Profiling Identifies Protein Disulfide-Isomerases as Target Proteins of the Volatile Salinilactones.

The salinilactones, volatile marine natural products secreted from Salinispora arenicola, feature a unique [3.1.0]-lactone ring system and cytotoxic activities through a hitherto unknown mechanism. To find their molecular target, an activity-based protein profiling with a salinilactone-derived probe is applied that disclosed the protein disulfide-isomerases (PDIs) as the dominant mammalian targets of salinilactones, and thioredoxin (TRX1) as secondary target. The inhibition of protein disulfide-isomerase A1 (PDIA1) and TRX1 is confirmed by biochemical assays with recombinant proteins, showing that (1S,5R)-salinilactone B is more potent than its (1R,5S)-configured enantiomer. The salinilactones bound covalently to C53 and C397, the catalytically active cysteines of the isoform PDIA1 according to tandem mass spectrometry. Reactions with a model substrate demonstrated that the cyclopropyl group is opened by an attack of the thiol at C6. Fluorophore labeling experiments showed the cell permeability of a salinilactone-BODIPY (dipyrrometheneboron difluoride) conjugate and its co-localization with PDIs in the endoplasmic reticulum. The study is one of the first to pinpoint a molecular target for a volatile microbial natural product, and it demonstrates that salinilactones can achieve high selectivity despite their small size and intrinsic reactivity.

Fluorescence microscopy shadow imaging for neuroscience.

Fluorescence microscopy remains one of the single most widely applied experimental approaches in neuroscience and beyond and is continuously evolving to make it easier and more versatile. The success of the approach is based on synergistic developments in imaging technologies and fluorophore labeling strategies that have allowed it to greatly diversify and be used across preparations for addressing structure as well as function. Yet, while targeted labeling strategies are a key strength of fluorescence microscopy, they reciprocally impose general limitations on the possible types of experiments and analyses. One recent development that overcomes some of these limitations is fluorescence microscopy shadow imaging, where membrane-bound cellular structures remain unlabeled while the surrounding extracellular space is made to fluoresce to provide a negative contrast shadow image. When based on super-resolution STED microscopy, the technique in effect provides a positive image of the extracellular space geometry and entire neuropil in the field of view. Other noteworthy advantages include the near elimination of the adverse effects of photobleaching and toxicity in live imaging, exhaustive and homogeneous labeling across the preparation, and the ability to apply and adjust the label intensity on the fly. Shadow imaging is gaining popularity and has been applied on its own or combined with conventional positive labeling to visualize cells and synaptic proteins in their parenchymal context. Here, we highlight the inherent limitations of fluorescence microscopy and conventional labeling and contrast these against the pros and cons of recent shadow imaging approaches. Our aim is to describe the brief history and current trajectory of the shadow imaging technique in the neuroscience field, and to draw attention to its ease of application and versatility.

Self-quenched fluorophore labels for DNA-PAINT and STED microscopy

Self-quenched fluorophore labels for DNA-PAINT and STED microscopy

Multitarget Fluorescence In Situ Hybridization Diagnostic Applications in Tumors.

Multitarget fluorescence in situ hybridization (mFISH) is a technique that allows the detection of multiple target sequences on the same sample using spectrally distinct fluorophore labels. The mFISH approach is currently a useful assay in the oncologic field for the detection of predictive, prognostic, and diagnostic biomarkers. In this chapter, we summarize the application of mFISH in the identification of target genetic aberrations in formalin-fixed, paraffin-embedded (FFPE) tissue samples of several tumor types. We discuss the mFISH protocols in FFPE samples, the innovative multitarget probes used, and the critical issues related to their interpretation.

High-throughput assay exploiting disorder-to-order conformational switches: application to the proteasomal Rpn10:E6AP complex.

Conformational switching is pervasively driven by protein interactions, particularly for intrinsically disordered binding partners. We developed a dually orthogonal fluorescence-based assay to monitor such events, exploiting environmentally sensitive fluorophores. This assay is applied to E3 ligase E6AP, as its AZUL domain induces a disorder-to-order switch in an intrinsically disordered region of the proteasome, the so-named Rpn10 AZUL-binding domain (RAZUL). By testing various fluorophores, we developed an assay appropriate for high-throughput screening of Rpn10:E6AP-disrupting ligands. We found distinct positions in RAZUL for fluorophore labeling with either acrylodan or Atto610, which had disparate spectral responses to E6AP binding. E6AP caused a hypsochromic shift with increased fluorescence of acrylodan-RAZUL while decreasing fluorescence intensity of Atto610-RAZUL. Combining RAZUL labeled with either acrylodan or Atto610 into a common sample achieved robust and orthogonal measurement of the E6AP-induced conformational switch. This approach is generally applicable to disorder-to-order (or vice versa) transitions mediated by molecular interactions.

Comprehensive Characterization of Evolution of Genomic Complexity By Structural Variant and Mutational Profiling in Myelodysplastic Syndrome Patients with Hypomethylating Agent Failure

Introduction MDS patients (pts) who develop resistance to hypomethylating agent therapy (HMA-F) have dismal outcomes. Genomic aberrations underlying progression to HMA-F remains poorly understood. By sequencing, studies have demonstrated acquisition of new mutations affecting transcription factors such as FLT3 and RAS-MAPK pathway genes in a subset of cases. Until now, evolution of genomic complexity in HMA-F by acquisition of new structural variants (SVs) has not been systematically investigated. This is due to lack of genome-wide technologies to quantitatively identify all types of SVs at a gene/exon-level resolution (deletions/insertions/inversions/fusions etc..). In this study, we optical genome mapping (OGM), a novel, non-sequencing-based approach for accurate high-resolution SV detection, combined with ultra-deep coverage targeted NGS to comprehensively analyze genome-wide changes affecting both SVs and somatic mutations using paired baseline and progression samples from 21 MDS pts who developed HMA-F. Methods We identified all MDS pts with sufficient (1.5 million) cryopreserved, viable bone marrow (BM) mononuclear cells at both diagnosis (baseline) and HMA-F. All pts underwent BM morphologic exam, traditional banding analysis (TBA), and 81-gene targeted NGS panel (2500X coverage; LOD: 2% VAF; myeloid genes). We excluded pts with AML-defining abnormalities by standard-of-care work-up (per WHO 2022 criteria) and CMML pts. OGM was performed using standard procedures: extraction of ultra-long DNA molecules, sequence-specific fluorophore labeling and stain, followed by loading on to nanochannels and imaging (Saphyr; Bionano Genomics). Rare Variant Pipeline was used for detection of clinically significant SVs [minimum 300X coverage; 5000 bp size cut-off). We used bidirectional paired genome-wide analysis for identifying unique SVs seen exclusively at either baseline or HMA-F. Results There were 21 pts [11 men; 10 women; median age: 73 (43-89)] with available cryopreserved cells for comprehensive mutational and SV profiling. Median BM blast% was 5 (1-18). The median time to HMA failure was 11 (6-21) months. All pts had at least 1 genomic aberration: 14 (66%) by TBA and 20 (95%) by NGS (median: 3 mutations per pt.). Seven (35%) pts had normal karyotype (NK), 6 (30%) pts had complex karyotype (CK). Most frequent gene mutations included TET2, ASXL1 TP53 [each seen in 7 pts, 33%], SRSF2 [6 (30%)], DNMT3A, RUNX1 and STAG2 (5 pts each, 25%). Baseline OGM identified multiple clinically significant SVs, 6 of which, seen across 4 (19%) pts, were cryptic by TBA. These included CBFB::MYH1 and KMT2A partial tandem duplication. OGM at HMA-F showed increased genomic complexity compared to baseline in 9 (43%) pts. Paired analysis comparing genome-wide SV burden at HMA-F with matched baseline sample revealed 306 new unique SVs at HMA-F not seen at baseline ( Fig 1). These included 30 deletions (8 pts), duplications (6 pts), inversions, intrachromosomal and intrachromosomal translocations (5 pts each). Recurrent aberrations included segmental deletions of chr 17, 1, 7, 9, 12 and 21, affecting ETV6, CDKN1B, IKZF1, RUNX1, FHIT and HOXA11 genes, exon-spanning duplication in RUNX1, and FANCA::NF1 and YTHDF3-SETBP1 fusions. Three (14%) pts (2 with CK, 1 with NK), showed a cataclysmic increase in the numbers of SVs (translocations/ inversions) indicative of chromoanagenesis ( Fig 2). In contrast, there were 29 SVs seen exclusively at baseline and not detected at HMA-F [intrachromosomal translocations affecting chr 3 ( FOXP1) and chr 7 ( CUX1), low-level IGH::BCL2, deletions and insertions]. Sequential NGS and TBA was available in 20 pts. At HMA-F, 13 (62%) pts developed additional mutations, most frequent in NRAS, followed by TP53, TET2, CSF3R, KIT, second mutation in CEBPA, DDX41 and WT1 genes. The median number of mutations per pt. was 4. The highest increase in SV burden by OGM was noted in pts who did not acquire new mutations by NGS at HMA-F (5/7; 71%). Seven (33%) pts showed cytogenetic clonal evolution at HMA-F by TBA. OGM identified new SVs at HMA-F in 2 (50%) pts who did not show clonal evolution by NGS or TBA. Conclusions HMA-F MDS is characterized by increased genomic complexity by acquisition of new somatic mutations (NGS) and SVs (OGM), highlighting their complementary role in MDS progression. OGM further highlighted cataclysmic genomic changes by chromoanagenesis in a subset of MDS pts at HMA-F.

Purification free N-glycan analysis by capillary zone electrophoresis: Hunt for the lost glycans

Capillary gel electrophoresis is a widely used method for rapid separation of fluorophore labeled carbohydrates. Even though, many publications conferred about this popular technique, no report yet investigated the possible sample losses during the purification process of the fluorophore labeling reaction mixture. In the present work, normal polarity capillary zone electrophoresis separation mode was applied to take advantage of the opposite migration directions of the electroosmotic flow and the negatively charged sample components using Tris-hexanoic acid running buffer at basic pH. For purification free oligosaccharide analysis, the separation parameters were designed in such a way that the triple charged labeling reagent of aminopyrenetrisulfonate (APTS) could not enter the separation capillary in contrary to the labeled sample components of interest, therefore, the APTS did not have to be removed before analysis. The method was used to show electrophoretic profile differences possibly caused by the cleanup process that was immediately apparent by comparing the electropherograms of the purified and non-purified APTS labeled maltooligosaccharides. Furthermore, qualitative and quantitative N-glycosylation profile alterations were revealed during CZE separation of the fluorophore labeling reaction mixtures before and after purification along with the analysis of the consecutively used washing solutions for the well characterized standard glycoproteins of IgG, ribonuclease B and fetuin.

Amplification-free microRNA profiling with femtomolar sensitivity on a plasmonic enhanced fluorescence nano-chip

MicroRNAs (miRNAs) are a class of small, non-coding RNA molecules involved in the regulation of gene expression, thus considered as promising biomarkers for cancer, cardiovascular diseases, neurodegenerative diseases, etc. However, quantitative analysis of miRNAs faces challenges owing to their high homology, small size & ultra-low abundance, and disease occurrence is often related to abnormal expression of multiple miRNAs where method for parallel miRNAs analysis is required. In this work, multiplexed analysis of miRNAs was established on a plasmonic nano-chip capable of fluorescence enhancement in the near-infrared region. Combined with polyadenylation at the hydroxyl terminate of target miRNA to afford abundant sites for fluorophore labeling, our assay achieved amplification-free detection of miRNAs from nM to fM with the limit of detection down to ca. 5 fM. A miRNA panel was constructed to detect 10 miRNAs differentially expressed in MCF-7 and A549 cell lines and validated with qRT-PCR, demonstrating the practical application of this method. This scalable platform can be customized for different miRNA panels, facilitating multiple miRNA profiling for various diseases.

Time-resolved fluorescence anisotropy with Atto 488-labeled phytochrome Agp1 from Agrobacterium fabrum.

Phytochromes are photoreceptor proteins with a bilin chromophore that undergo photoconversion between two spectrally different forms, Pr and Pfr. Three domains, termed PAS, GAF, and PHY domains, constitute the N-terminal photosensory chromophore module (PCM); the C-terminus is often a histidine kinase module. In the Agrobacterium fabrum phytochrome Agp1, the autophosphorylation activity of the histidine kinase is high in the Pr and low in the Pfr form. Crystal structure analyses of PCMs suggest flexibility around position 308 in the Pr but not in the Pfr form. Here, we performed time-resolved fluorescence anisotropy measurements with different Agp1 mutants, each with a single cysteine residue at various positions. The fluorophore label Atto-488 was attached to each mutant, and time-resolved fluorescence anisotropy was measured in the Pr and Pfr forms. Fluorescence anisotropy curves were fitted with biexponential functions. Differences in the amplitude A2 of the second component between the PCM and the full-length variant indicate a mechanical coupling between position 362 and the histidine kinase. Pr-to-Pfr photoconversion induced no significant changes in the time constant t2 at any position. An intermediate t2 value at position 295, which is located in a compact environment, suggests flexibility around the nearby position 308 in Pr and in Pfr.

One pattern analysis (OPA) for the quantitative determination of protein interactions in plant cells

BackgroundA commonly used approach to study the interaction of two proteins of interest (POIs) in vivo is measuring Förster Resonance Energy Transfer (FRET). This requires the expression of the two POIs fused to two fluorescent proteins that function as a FRET pair. A precise way to record FRET is Fluorescence Lifetime IMaging (FLIM) which generates quantitative data that, in principle, can be used to resolve both complex structure and protein affinities. However, this potential resolution is often lost in many experimental approaches. Here we introduce a novel tool for FLIM data analysis of multiexponential decaying donor fluorophores, one pattern analysis (OPA), which allows to obtain information about protein affinity and complex arrangement by extracting the relative amplitude of the FRET component and the FRET transfer efficiency from other FRET parameters.ResultsAs a proof of concept for OPA, we used FLIM-FRET, or FLIM-FRET in combination with BiFC to reassess the dimerization and tetramerization properties of known interacting MADS-domain transcription factors in Nicotiana benthamiana leaf cells and Arabidopsis thaliana flowers. Using the OPA tool and by extracting protein BINDING efficiencies from FRET parameters to dissect MADS-domain protein interactions in vivo in transient N. benthamiana experiments, we could show that MADS-domain proteins display similar proximities within dimeric or tetrameric complexes but bind with variable affinities. By combining FLIM with BiFC, we were able to identify SEPALLATA3 as a mediator for tetramerization between the other MADS-domain factors. OPA also revealed that in vivo expression from native promoters at low levels in Arabidopsis flower meristems, makes in situ complex formation of MADS-domain proteins barely detectable.ConclusionsWe conclude that MADS-domain protein interactions are transient in situ and may involve additional, so far unknown interaction mediators. We conclude that OPA can be used to separate protein binding from information about proximity and orientation of the interacting proteins in their complexes. Visualization of individual protein interactions within the underlying interaction networks in the native environment is still restrained if expression levels are low and will require continuous improvements in fluorophore labelling, instrumentation set-ups and analysis tools.

Development of Highly Dense Material-Specific Fluorophore Labeling Method on Silicon-Based Semiconductor Materials for Three-Dimensional Multicolor Super-Resolution Fluorescence Imaging

Development of Highly Dense Material-Specific Fluorophore Labeling Method on Silicon-Based Semiconductor Materials for Three-Dimensional Multicolor Super-Resolution Fluorescence Imaging

Enzymatic reaction modulated DNA assembly on graphitic carbon nitride nanosheets for sensitive fluorescence detection of acetylcholinesterase activity and inhibition.

A novel fluorescent strategy has been developed by using anenzymatic reaction modulated DNA assembly on graphitic carbon nitride nanosheets (CNNS) for the detection of acetylcholinesterase (AChE) activity and its inhibitors. Thetwo-dimensional and ultrathin-layer CNNS-material was successfully synthesized through a chemical oxidation and ultrasound exfoliation method. Because of its excellent adsorption selectivity to ssDNA over dsDNA and superior quenching ability toward the fluorophore labels, CNNS were employed to construct a sensitive fluorescence sensing platform for the detection of AChE activity and inhibition. The detection was based on enzymatic reaction modulated DNA assembly on CNNS, which involved the specific AChE-catalyzed reaction-mediated DNA/Hg2+ conformational change and subsequent signal transduction and amplification via hybridization chain reaction (HCR). Under the excitation at 485 nm, the fluorescence signal from 500 to 650 nm (λmax = 518 nm) of the developed sensing system was gradually increased with increasing concentration of AChE. The quantitative determinationrange of AChE is from 0.02 to 1 mU/mL and the detection limit was 0.006 mU/mL. The developed strategy was successfully applied to the assay of AChE in human serum samples, and can also be used to effectively screen AChE inhibitors, showing great promise providinga robust and effective platform for AChE-related diagnosis, drug screening, and therapy.