Fluorescence Resonance Energy Transfer Approach Research Articles

Mechanistic Innovations in Fluorescent Chemosensors for Detecting Toxic Ions: PET, ICT, ESIPT, FRET and AIE Approaches.

Fluorescent chemosensors have become vital tools for detecting toxic ions due to their exceptional sensitivity, selectivity, and rapid response times. These sensors function through various mechanisms, each providing unique advantages for specific applications. This review offers a comprehensive overview of the mechanistic innovations in fluorescent chemosensors, emphasizing five key approaches: Photoinduced Electron Transfer (PET), Fluorescence Resonance Energy Transfer (FRET), Intramolecular Charge Transfer (ICT), Aggregation-Induced Emission (AIE), and Excited-State Intramolecular Proton Transfer (ESIPT). We highlight the substantial progress made in developing these chemosensors, discussing their design principles, sensing mechanisms, and practical applications, with a particular focus on their use in detecting toxic ions relevant to environmental and biological contexts.

A reliable and selective ratiometric sensing probe for fluorometric determination of P2O74− based on AIE of GSH@CuNCs-assisted by Al-N@CQDs

In this study, a novel composite material was developed for the ratiometric detection of pyrophosphate anion (P2O74−). This composite consisted of Al and nitrogen co-doped carbon dots (Al-N@CQDs) and glutathione-capped copper nanoclusters (GSH@CuNCs). The Al-N@CQDs component, with its high reserved coordination capacity of Al3+, induced the non-luminescent behavior of GSH@CuNCs, resulting in an aggregation-induced emission (AIE) effect. The hybrid material (Al-N@CQDs/GSH@CuNCs) exhibited dual-emission signals at 620 nm and 450 nm after integrating the two independent materials utilizing the AIE effect and the fluorescence resonance energy transfer (FRET) approach. This approach represents the first utilization of this composite for ratiometric detection. Nevertheless, upon the addition of P2O74−, the AIE and FRET processes were hindered due to the higher coordination interaction of Al3+ towards P2O74− compared to the amino/carboxyl groups on Al-N@CQDs. This successful interference of the AIE and FRET processes allowed for the effective estimation of P2O74−. The response ratio (F450/F620) increased with increasing the concentration of P2O74− in the range of 0.035–160 µM, with an impressive detection limit of 0.012 µM. This innovative approach of utilizing hybrid CQDs/thiolate-capped nanoclusters as a ratiometric fluorescent sensor for analytical applications introduces new possibilities in the field. The as-fabricated system was successfully applied to detect P2O74− in different real samples such as water, serum, and urine samples with acceptable results.

Methods to investigate nucleosome structure and dynamics with single-molecule FRET.

The nucleosome is the fundamental building block of chromatin. Changes taking place at the nucleosome level are the molecular basis of chromatin transactions with various enzymes and factors. These changes are directly and indirectly regulated by chromatin modifications such as DNA methylation and histone post-translational modifications including acetylation, methylation, and ubiquitylation. Nucleosomal changes are often stochastic, unsynchronized, and heterogeneous, making it very difficult to monitor with traditional ensemble averaging methods. Diverse single-molecule fluorescence approaches have been employed to investigate the structure and structural changes of the nucleosome in the context of its interactions with various enzymes such as RNA Polymerase II, histone chaperones, transcription factors, and chromatin remodelers. We utilize diverse single-molecule fluorescence methods to study the nucleosomal changes accompanying these processes, elucidate the kinetics of these processes, and eventually learn the implications of various chromatin modifications in directly regulating these processes. The methods include two- and three-color single-molecule fluorescence resonance energy transfer (FRET), single-molecule fluorescence correlation spectroscopy, and fluorescence (co-)localization. Here we report the details of the two- and three-color single-molecule FRET methods we currently use. This report will help researchers design their single-molecule FRET approaches to investigating chromatin regulation at the nucleosome level.

Comparison of intensity versus lifetime-based approaches for quantitative in vivo FRET imaging.

Fluorescence Resonance Energy Transfer (FRET) enables measurement of the distance separating two light-sensitive molecules. The method has been employed in countless biological applications to monitor inter and intra-molecular interactions and conformations in the 1-10 nm range. Importantly, FRET measurements can be performed with both ratiometric intensity FRET (ratiometric FRET) and fluorescence lifetime imaging-based FRET (FLI-FRET) approaches. Although FRET has long been used for in vitro microscopy, using either the intensity- or lifetime-based approaches, it has recently been extended to in vivo application, for instance to quantify drug-target engagement in animal models of cancer. The in vivo method had thus far only been demonstrated using macroscopic FLI-FRET (MFLI-FRET). Herein, we present a comparison of FRET quantification in vivo using either ratiometric FRET (3-cube, IVIS system) and MFLI-FRET (time-resolved ICCD system) in order to demonstrate the superiority of the latter for in vivo applications.

Discrete RNA-DNA hybrid cleavage by the EXD2 exonuclease pinpoints two rate-limiting steps.

EXD2 is a recently identified exonuclease that cleaves RNA and DNA in double-stranded (ds) forms. It thus serves as a model system for investigating the similarities and discrepancies between exoribonuclease and exodeoxyribonuclease activities and for understanding the nucleic acid (NA) unwinding-degradation coordination of an exonuclease. Here, using a single-molecule fluorescence resonance energy transfer (smFRET) approach, we show that despite stable binding to both substrates, EXD2 barely cleaves dsDNA and yet displays both exoribonuclease and exodeoxyribonuclease activities toward RNA-DNA hybrids with a cleavage preference for RNA. Unexpectedly, EXD2-mediated hybrid cleavage proceeds in a discrete stepwise pattern, wherein a sudden 4-bp duplex unwinding increment and the subsequent dwell constitute a complete hydrolysis cycle. The relatively weak exodeoxyribonuclease activity of EXD2 partially originates from frequent hybrid rewinding. Importantly, kinetic analysis and comparison of the dwell times under varied conditions reveal two rate-limiting steps of hybrid unwinding and nucleotide excision. Overall, our findings help better understand the cellular functions of EXD2, and the cyclic coupling between duplex unwinding and exonucleolytic degradation may be generalizable to other exonucleases.

An Insight into Animal Glutamate Receptors Homolog of Arabidopsis thaliana and Their Potential Applications-A Review.

Most excitatory impulses received by neurons are mediated by ionotropic glutamate receptors (iGluRs). These receptors are located at the apex and play an important role in memory, neuronal development, and synaptic plasticity. These receptors are ligand-dependent ion channels that allow a wide range of cations to pass through. Glutamate, a neurotransmitter, activates three central ionotropic receptors: N-methyl-D-aspartic acid (NMDA), -amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA), and kainic acid (KA). According to the available research, excessive glutamate release causes neuronal cell death and promotes neurodegenerative disorders. Arabidopsis thaliana contains 20 glutamate receptor genes (AtGluR) comparable to the human ionotropic glutamate (iGluRs) receptor. Many studies have proved that AtGL-rec genes are involved in a number of plant growth and physiological activities, such as in the germination of seeds, roots, abiotic and biotic stress, and cell signaling, which clarify the place of these genes in plant biology. In spite of these, the iGluRs, Arabidopsis glutamate receptors (AtGluR), is associated with the ligand binding activity, which confirms the evolutionary relationship between animal and plant glutamate receptors. Along with the above activities, the impact of mammalian agonists and antagonists on Arabidopsis suggests a correlation between plant and animal glutamate receptors. In addition, these glutamate receptors (plant/animal) are being utilized for the early detection of neurogenerative diseases using the fluorescence resonance energy transfer (FRET) approach. However, a number of scientific laboratories and institutes are consistently working on glutamate receptors with different aspects. Currently, we are also focusing on Arabidopsis glutamate receptors. The current review is focused on updating knowledge on AtGluR genes, their evolution, functions, and expression, and as well as in comparison with iGluRs. Furthermore, a high throughput approach based on FRET nanosensors developed for understanding neurotransmitter signaling in animals and plants via glutamate receptors has been discussed. The updated information will aid in the future comprehension of the complex molecular dynamics of glutamate receptors and the exploration of new facts in plant/animal biology.

Cholesterol inhibits human voltage-gated proton channel hHv1

Although human sperm is morphologically mature in the epididymis, it cannot fertilize eggs before capacitation. Cholesterol efflux from the sperm plasma membrane is a key molecular event essential for cytoplasmic alkalinization and hyperactivation, but the underlying mechanism remains unclear. The human voltage-gated proton (hHv1) channel functions as an acid extruder to regulate intracellular pHs of many cell types, including sperm. Aside from voltage and pH, Hv channels are also regulated by distinct ligands, such as Zn2+ and albumin. In the present work, we identified cholesterol as an inhibitory ligand of the hHv1 channel and further investigated the underlying mechanism using the single-molecule fluorescence resonance energy transfer (smFRET) approach. Our results indicated that cholesterol inhibits the hHv1 channel by stabilizing the voltage-sensing S4 segment at resting conformations, a similar mechanism also utilized by Zn2+. Our results suggested that the S4 segment is the central gating machinery in the hHv1 channel, on which voltage and distinct ligands are converged to regulate channel function. Identification of membrane cholesterol as an inhibitory ligand provides a mechanism by which the hHv1 channel regulates fertilization by linking the cholesterol efflux with cytoplasmic alkalinization, a change that triggers calcium influx through the CatSper channel. These events finally lead to hyperactivation, a remarkable change in the mobility pattern indicating fertilization competence of human sperm.

Interaction of human CRX and NRL in live HEK293T cells measured using fluorescence resonance energy transfer (FRET)

CRX and NRL are retina-specific transcription factors that control rod photoreceptor differentiation and synergistically activate rod phototransduction gene expression. Previous experiments showed they interact in vitro and in yeast two-hybrid assays. Here, we examined CRX-NRL interaction in live HEK293T cells using two fluorescence resonance energy transfer (FRET) approaches: confocal microscopy and flow cytometry (FC-FRET). FC-FRET can provide measurements from many cells having wide donor–acceptor expression ranges. FRET efficiencies were calibrated with a series of donor (EGFP)-acceptor (mCherry) fusion proteins separated with linkers between 6–45 amino acids. CRX and NRL were fused at either terminus with EGFP or mCherry to create fluorescent proteins, and all combinations were tested in transiently transfected cells. FRET signals between CRX or NRL homo-pairs were highest with both fluorophores fused to the DNA binding domains (DBD), lower with both fused to the activation domains (AD), and not significant when fused on opposite termini. NRL had stronger FRET signals than CRX. A significant FRET signal between CRX and NRL hetero-pairs was detected when donor was fused to the CRX DNA binding domain and the acceptor fused to the NRL activation domain. FRET signals increased with CRX or NRL expression levels at a rate much higher than expected for collisional FRET alone. Together, our results show the formation of CRX-NRL complexes in live HEK293T cells that are close enough for FRET.

A direct interaction between RhoGDIα/Tau alleviates hyperphosphorylation of Tau in Alzheimer's disease and vascular dementia.

RhoGDIα is an inhibitor of RhoGDP dissociation that involves in Aβ metabolism and NFTs production in Alzheimer's disease (AD) by regulating of RhoGTP enzyme activity. Our previous research revealed that RhoGDIα, as the target of Polygala saponin (Sen), might alleviate apoptosis of the nerve cells caused by hypoxia/reoxygenation (H/R). To further clarify the role of RhoGDIα in the generation of NFTs, we explored the relationship between RhoGDIα and Tau. We found out that RhoGDIα and Tau can bind with each other and interact by using coimmunoprecipitation (Co-IP) and GST pulldown methods in vitro. This RhoGDIα-Tau partnership was further verified by using immunofluorescence colocalization and fluorescence resonance energy transfer (FRET) approaches in PC12 cells. Using the RNA interference (RNAi) technique, we found that the RhoGDIα may be involved in an upstream signaling pathway for Tau. Subsequently, in Aβ25-35- and H/R-induced PC12 cells, forced expression of RhoGDIα via cDNA plasmid transfection was found to reduce the hyperphosphorylation of Tau, augment the expression of bcl-2 protein, and inhibit the expression of Bax protein (reducing the Bax/bcl-2 ratio) and the activity of caspase-3. In mouse AD and VaD models, forced expression of RhoGDIα via injection of a viral vector (pAAV-EGFP-RhoGDIα) into the lateral ventricle of the brain alleviated the pathological symptoms of AD and VaD. Finally, GST pulldown confirmed that the binding sites on RhoGDIα for Tau were located in the range of the ΔC33 fragment (aa 1-33). These results indicate that RhoGDIα is involved in the phosphorylation of Tau and apoptosis in AD and VaD. Overexpression of RhoGDIα can inhibit the generation of NFTs and delay the progress of these two types of dementia.

Characterization of TERRA 9 repeat G4-binding RHAU peptides by Fluorescence Resonance Energy Transfer

Introduction: TERRA (telomeric repeat-containing RNA) is a G-quadruplex (G4) noncoding RNA that plays a part in cancer and aging. Here, we report the use of a fluorescence resonance energy transfer (FRET) approach to explore the binding mode of TERRA 9 repeat G4-binding RHAU peptides. Methods: RHAU peptide was genetically incorporated with the fluorescent protein CFP/YFP, resulting in the generation of the fluorescent protein FRET and RHAU55-CFP/RHAU55-YFP. Results: A strong hetero-FRET was observed when TERRA 9 repeats were added to the RHAU55-CFP/RHAU55-YFP mixture, indicating that TERRA 9 repeats could form G4 structures and simultaneously bind multiple proteins fused with the RHAU peptide. Conclusion: This provides a useful approach for regulating the telomere elongation process by a complex of TERRA and functional proteins fused with the RHAU peptide.

METTL3 is a key regulator of milk synthesis in mammary epithelial cells

The enzyme m6 A methyltransferase-like 3 (METTL3) catalyzes N6 -methyladenosine (m6 A) modification in eukaryotic messenger RNAs (mRNAs). However, the physiological function and molecular mechanism of METTL3 in mammalian cells have not been fully understood. Here we showed that METTL3 was highly expressed in mouse mammary gland of the lactation period. METTL3 was located in the nucleus of bovine mammary epithelial cells (MECs), and methionine (Met) and β-estrodial (E2) upregulated METTL3 protein level. METTL3 knockdown decreased milk protein and fat synthesis, whereas its overexpression had the opposite effects. METTL3 overexpression stimulated mRNA expression and protein phosphorylation of the mechanistic target of rapamycin (mTOR) and mRNA and protein expression of sterol regulatory element binding protein 1 (SREBP1), whereas METTL3 knockdown blocked the stimulatory effects of Met and E2 on these processes. Furthermore, METTL3 overexpression led to increased mRNA m6 A methylation of mTOR and SREBP1, whereas METTL3 knockdown suppressed the stimulatory effects of Met and E2 on these processes. The interaction between METTL3 and glycyl-tRNA synthetase (GlyRS) was confirmed by Co-immunoprecipitation and fluorescence resonance energy transfer approaches, and colocalization observation further showed that Met and E2 treatment increased this interaction. GlyRS knockdown abolished METTL3 protein levels upregulated by Met and E2, and METTL3 knockdown markedly decreased the effects of GlyRS overexpression on mTOR expression and phosphorylation and SREBP1 expression. In summary, we demonstrate that METTL3 is a key positive regulator of Met and E2-stimulated and GlyRS-mediated mTOR and SREBP1 signaling pathways and milk protein and fat synthesis in mammary epithelial cells.

First passage time study of DNA strand displacement

DNA strand displacement, in which a single-stranded nucleic acid invades a DNA duplex, is pervasive in genomic processes and DNA engineering applications. The kinetics of strand displacement have been studied in bulk; however, the kinetics of the underlying strand exchange were obfuscated by a slow bimolecular association step. Here, we use a novel single-molecule fluorescence resonance energy transfer approach termed the “fission” assay to obtain the full distribution of first passage times of unimolecular strand displacement. At a frame time of 4.4 ms, the first passage time distribution for a 14-nucleotide displacement domain exhibited a nearly monotonic decay with little delay. Among the eight different sequences we tested, the mean displacement time was on average 35 ms and varied by up to a factor of 13. The measured displacement kinetics also varied between complementary invaders and between RNA and DNA invaders of the same base sequence, except for T → U substitution. However, displacement times were largely insensitive to the monovalent salt concentration in the range of 0.25–1 M. Using a one-dimensional random walk model, we infer that the single-step displacement time is in the range of ∼30–300 μs, depending on the base identity. The framework presented here is broadly applicable to the kinetic analysis of multistep processes investigated at the single-molecule level.

Unveiling the N-Terminal Homodimerization of BCL11B by Hybrid Solvent Replica-Exchange Simulations.

Transcription factors play a crucial role in regulating biological processes such as cell growth, differentiation, organ development and cellular signaling. Within this group, proteins equipped with zinc finger motifs (ZFs) represent the largest family of sequence-specific DNA-binding transcription regulators. Numerous studies have proven the fundamental role of BCL11B for a variety of tissues and organs such as central nervous system, T cells, skin, teeth, and mammary glands. In a previous work we identified a novel atypical zinc finger domain (CCHC-ZF) which serves as a dimerization interface of BCL11B. This domain and formation of the dimer were shown to be critically important for efficient regulation of the BCL11B target genes and could therefore represent a promising target for novel drug therapies. Here, we report the structural basis for BCL11B–BCL11B interaction mediated by the N-terminal ZF domain. By combining structure prediction algorithms, enhanced sampling molecular dynamics and fluorescence resonance energy transfer (FRET) approaches, we identified amino acid residues indispensable for the formation of the single ZF domain and directly involved in forming the dimer interface. These findings not only provide deep insight into how BCL11B acquires its active structure but also represent an important step towards rational design or selection of potential inhibitors.

Measuring Lipid Transfer Protein Activity Using Bicelle-Dilution Model Membranes.

In vitro assessment of lipid intermembrane transfer activity by cellular proteins typically involves measurement of either radiolabeled or fluorescently labeled lipid trafficking between vesicle model membranes. Use of bilayer vesicles in lipid transfer assays usually comes with inherent challenges because of complexities associated with the preparation of vesicles and their rather short "shelf life". Such issues necessitate the laborious task of fresh vesicle preparation to achieve lipid transfer assays of high quality, precision, and reproducibility. To overcome these limitations, we have assessed model membrane generation by bicelle dilution for monitoring the transfer rates and specificity of various BODIPY-labeled sphingolipids by different glycolipid transfer protein (GLTP) superfamily members using a sensitive fluorescence resonance energy transfer approach. Robust, protein-selective sphingolipid transfer is observed using donor and acceptor model membranes generated by dilution of 0.5 q-value mixtures. The sphingolipid transfer rates are comparable to those observed between small bilayer vesicles produced by sonication or ethanol injection. Among the notable advantages of using bicelle-generated model membranes are (i) easy and straightforward preparation by means that avoid lipid fluorophore degradation and (ii) long "shelf life" after production (≥6 days) and resilience to freeze-thaw storage. The bicelle-dilution-based assay is sufficiently robust, sensitive, and stable for application, not only to purified LTPs but also for LTP activity detection in crude cytosolic fractions of cell homogenates.

Real-time monitoring of glutathione in living cells using genetically encoded FRET-based ratiometric nanosensor

Reduced glutathione (GSH) level inside the cell is a critical determinant for cell viability. The level of GSH varies across the cells, tissues and environmental conditions. However, our current understanding of physiological and pathological GSH changes at high spatial and temporal resolution is limited due to non-availability of practicable GSH-detection methods. In order to measure GSH at real-time, a ratiometric genetically encoded nanosensor was developed using fluorescent proteins and fluorescence resonance energy transfer (FRET) approach. The construction of the sensor involved the introduction of GSH binding protein (YliB) as a sensory domain between cyan fluorescent protein (CFP; FRET donor) and yellow fluorescent protein (YFP; FRET acceptor). The developed sensor, named as FLIP-G (Fluorescence Indicator Protein for Glutathione) was able to measure the GSH level under in vitro and in vivo conditions. When the purified FLIP-G was titrated with different concentrations of GSH, the FRET ratio increased with increase in GSH-concentration. The sensor was found to be specific for GSH and also stable to changes in pH. Moreover, in live bacterial cells, the constructed sensor enabled the real-time quantification of cytosolic GSH that is controlled by the oxidative stress level. When expressed in yeast cells, FRET ratio increased with the external supply of GSH to living cells. Therefore, as a valuable tool, the developed FLIP-G can monitor GSH level in living cells and also help in gaining new insights into GSH metabolism.

A highly selective “Turn On” fluorescent probe based on FRET mechanism for hydrogen sulfide detection in living cells

A novel class of fluorescent “turn on” probe based on the fluorescence resonance energy transfer (FRET) approach was designed, and successfully synthesized for the detection of hydrogen sulfide (H2S) both in vitro and in vivo. This new H2S responsive fluorescent probe was developed based on the basis of the dabsyl and fluorescein FRET system. In the presence of H2S, a fluorescence enhancement was markedly observed mainly due to the inhibition of the FRET process. This probe could quantitatively measure the level of H2S with the detection limit of 0.02 μM. Cell imaging results demonstrated the potential applicability of a dabsylfluorescein probe for H2S detection in living cells.

Salivary Total Protease Activity Based on a Broad-Spectrum Fluorescence Resonance Energy Transfer Approach to Monitor Induction and Resolution of Gingival Inflammation.

ObjectiveSalivary total protease and chitinase activities were measured by a broad-spectrum fluorescence resonance energy transfer approach as predictors of induction and resolution of gingival inflammation in healthy individuals by applying an experimental human gingivitis model.MethodsDental biofilm accumulated (21 days, Induction Phase) by omitting oral hygiene practices followed by a 2-week Resolution Phase to restore gingival health in an experimental gingivitis study. Plaque accumulation, as assessed by the Turesky Modification of the Quigley-Hein Plaque Index (TQHPI), and gingival inflammation, assessed using the Modified Gingival Index (MGI), scores were recorded and unstimulated saliva was collected weekly. Saliva was analysed for total protein, albumin, total protease activity and chitinase activity (n = 18).ResultsThe TQHPI and MGI scores, as well as total protease activity, increased until day 21. After re-establishment of oral hygiene, gingival inflammation levels returned to values similar to baseline (day 0). Levels of protease activity decreased significantly, but not to baseline values. Furthermore, ‘fast’ responders, who responded immediately to plaque, exhibited significantly higher proteolytic activity throughout the experimental course than ‘slow’ responders, who showed a lagged inflammatory response.ConclusionThe results indicate that differential inflammatory responses encompass inherent variations in total salivary proteolytic activities, which could be further utilised in contemporary diagnostic, prognostic and treatment modalities for periodontal diseases.

Iron-Dependent Trafficking of 5-Lipoxygenase and Impact on Human Macrophage Activation.

5-lipoxygenase (5-LOX) is a non-heme iron-containing dioxygenase expressed in immune cells that catalyzes the two initial steps in the biosynthesis of leukotrienes. It is well known that 5-LOX activation in innate immunity cells is related to different iron-associated pro-inflammatory disorders, including cancer, neurodegenerative diseases, and atherosclerosis. However, the molecular and cellular mechanism(s) underlying the interplay between iron and 5-LOX activation are largely unexplored. In this study, we investigated whether iron (in the form of Fe3+ and hemin) might modulate 5-LOX influencing its membrane binding, subcellular distribution, and functional activity. We proved by fluorescence resonance energy transfer approach that metal removal from the recombinant human 5-LOX, not only altered the catalytic activity of the enzyme, but also impaired its membrane-binding. To ascertain whether iron can modulate the subcellular distribution of 5-LOX in immune cells, we exposed THP-1 macrophages and human primary macrophages to exogenous iron. Cells exposed to increasing amounts of Fe3+ showed a redistribution (ranging from ~45 to 75%) of the cytosolic 5-LOX to the nuclear fraction. Accordingly, confocal microscopy revealed that acute exposure to extracellular Fe3+, as well as hemin, caused an overt increase in the nuclear fluorescence of 5-LOX, accompanied by a co-localization with the 5-LOX activating protein (FLAP) both in THP-1 macrophages and human macrophages. The functional relevance of iron overloading was demonstrated by a marked induction of the expression of interleukin-6 in iron-treated macrophages. Importantly, pre-treatment of cells with the iron-chelating agent deferoxamine completely abolished the hemin-dependent translocation of 5-LOX to the nuclear fraction, and significantly reverted its effect on interleukin-6 overexpression. These results suggest that exogenous iron modulates the biological activity of 5-LOX in macrophages by increasing its ability to bind to nuclear membranes, further supporting a role for iron in inflammation-based diseases where its homeostasis is altered and suggesting further evidence of risks related to iron overload.

A NIR-triggered gatekeeper of supramolecular conjugated unimicelles with two-photon absorption for controlled drug release.

A near-infrared (NIR)-sensitive gated assembly of supramolecular conjugated unimicelles based on robust host-guest recognition between a β-cyclodextrin-grafted hyperbranched conjugated polymer and azobenzene-functionalized poly(ethylene glycol) was constructed. Utilized as a drug carrier, these unimicelles exhibited controlled drug release through the NIR-triggered photoisomerization of azobenzene in cancer cells via a two-photon excited fluorescence resonance energy transfer (TP-FRET) approach, leading to efficient cancer therapy.

Preparation of Plant Material for Analysis of Protein-Nucleic Acid Interactions by FRET-FLIM.

DNA-binding proteins are involved in the dynamic regulation of various cellular processes such as recombination, replication, and transcription. For investigating dynamic assembly and disassembly of molecular complexes in living cells, fluorescence microscopy represents a tremendous tool in biology. A fluorescence resonance energy transfer (FRET) approach coupled to fluorescence lifetime imaging microscopy (FLIM) has been used recently to monitor protein-DNA associations in plant cells. With this approach, the donor fluorophore is a GFP-tagged binding partner expressed in plant cells. A Sytox® Orange treatment converts nuclear nucleic acids to FRET acceptors. A decrease of GFP lifetime is due to FRET between donor and acceptor, indicating close association of the GFP binding partner and Sytox® Orange-stained DNA. In this chapter, we present a step-by-step protocol for the transient expression in N. benthamiana of GFP-tagged proteins and the fixation and permeabilization procedures used for the preparation of plant material aimed at detecting protein-nucleic acid interactions by FRET-FLIM measurements.