Resonance Energy Transfer Probes Research Articles

Specific FRET Probes Sensitive to Chitosan-Based Polymeric Micelles Formation, Drug-Loading, and Fine Structural Features.

Förster resonance energy transfer (FRET) probes are a promising tool for studying numerous biochemical processes. In this paper, we show the application of the FRET phenomenon to observe the micelle formation from surfactants, micelles self-assembling from chitosan grafted with fatty acid (oleic-OA, or lipoic-LA), cross-linking of SH groups in the micelle's core, and inclusion and release of the model drug cargo from the micelles. Using the carbodiimide approach, amphiphilic chitosan-based polymers with (1) SH groups, (2) crosslinked with S-S between polymer chains, and (3) without SH and S-S groups were synthesized, followed by characterization by FTIR and NMR spectroscopy. Two pairs of fluorophores were investigated: 4-methylumbelliferon-trimethylammoniocinnamate-rhodamine (MUTMAC-R6G) and fluorescein isothiocyanate-rhodamine (FITC-R6G). While FITC-R6G has been described before as an FRET-producing pair, for MUTMAC-R6G, this has not been described. R6G, in addition to being an acceptor fluorophore, also serves as a model cytostatic drug in drug-release experiments. As one could expect, in aqueous solution, FRET effect was poor, but when exposed to the micelles, both MUTMAC-R6G and FITC-R6G yielded a pronounced FRET effect. Most likely, the formation of micelles is accompanied by the forced convergence of fluorophores in the hydrophobic micelle core by a donor-to-acceptor distance (r) significantly closer than in the aqueous buffer solution, which was reflected in the increase in the FRET efficiency (E). Therefore, r(E) could be used as analytical signal of the micelle formation, including critical micelle concentration (CMC) and critical pre-micelle concentration (CPMC), yielding values in good agreement with the literature for similar systems. We found that the r-function provides analytically valuable information about the nature and mechanism of micelle formation. S-S crosslinking between polymer chains makes the micelle more compact and stable in the normal physiological conditions, but loosens in the glutathione-rich tumor microenvironment, which is considered as an efficient approach in targeted drug delivery. Indeed, we found that R6G, as a model cytostatic agent, is released from micelles with initial rate of 5%/h in a normal tissue microenvironment, but in a tumor microenvironment model (10 mM glutathione), the release of R6G from S-S stitched polymeric micelles increased up to 24%/h. Drug-loading capacity differed substantially: from 75-80% for nonstitched polymeric micelles to ~90% for S-S stitched micelles. Therefore, appropriate FRET probes can provide comprehensive information about the micellar system, thus helping to fine-tune the drug delivery system.

Highly selective detection of deoxyribonucleic acid in living cells using RecA-green fluorescent protein-single-stranded deoxyribonucleic acid filament fluorescence resonance energy transfer probe.

A fluorescence resonance energy transfer (FRET) method was developed for double-stranded deoxyribonucleic acid (dsDNA) detection in living cells using the RecA-GFP (green fluorescent protein) fusion protein filament. In brief, the thiol-modified single-stranded DNA (ssDNA) was attached to gold nanoparticles (AuNPs); on the contrary, the prepared RecA-GFP fusion protein interacted with ssDNA. Due to the FRET between AuNPs and RecA-GFP, fluorescence of RecA-GFP fusion protein was quenched. In the presence of homologous dsDNA, homologous recombination occurred to release RecA-GFP fusion protein. Thus, the fluorescence of RecA-GFP was recovered. The dsDNA concentration was detected using fluorescence intensity of RecA-GFP. Under optimal conditions, this method could detect dsDNA activity as low as 0.015 optical density (OD) Escherichia coli cells, with a wide linear range from 0.05 to 0.9 OD cells, and the regression equation was ΔF = 342.7c + 78.9, with a linear relationship coefficient of 0.9920. Therefore, it provided a promising approach for the selective detection of dsDNA in living cells for early clinical diagnosis of genetic diseases.

A highly sensitive fluorescent probe RN-NA reveals peroxynitrite as a novel biomarker for primary open angle glaucoma.

To directly quantify peroxynitrite (ONOO-) using a highly sensitive fluorescence resonance energy transfer probe RN-NA, investigate the association between ONOO- and primary open angle glaucoma (POAG), and clarify whether RN-NA could be used as a potential tool for POAG diagnosis. Plasma and aqueous humor (AH) samples were collected from POAG patients (n=100, age: 59.70±6.87y) and age-related cataract (ARC) patients (n=100, age: 61.15±4.60y) admitted to our hospital. Next, RN-NA was used to detect ONOO- in plasma and AH samples, and the relationship between ONOO- level and POAG was analyzed using binary logistic regression. Besides, Pearson correlation analysis was applied to characterize the correlation of the levels of ONOO- with the patients' age, intraocular pressure (IOP), and mean deviation of visual field testing. The ONOO- scavenger MnTMPyP was employed to treat the 3-morpholinosyndnomine (SIN-1)-induced ocular hypertension in mice (n=7, 6-8wk). Finally, the IOP and ONOO- in both eyes were measured 30min after the last drug treatment. ONOO- levels of AH and plasma were significantly higher in the POAG group than in the ARC group (P<0.01). Additionally, ONOO- levels were closely correlated with POAG in a binary logistic regression analysis [odds ratio (OR)=1.008, 95% confidence interval (CI): 1.002-1.013, P<0.01 for AH; OR=1.004, 95%CI: 1.002-1.006, P<0.001 for plasma]. Pearson correlation analysis showed that ONOO- levels in AH or plasma were positively associated with visual field defects (R=0.51, P<0.01 for AH; R=0.45, P<0.001 for plasma), and ONOO- levels in plasma and AH were correlated in the POAG group (R=0.69, P<0.001). However, administering MnTMPyP to mouse eyes reversed the elevated IOP caused by SIN-1 (P<0.05). ONOO- levels in AH and plasma, detected by RN-NA, are significantly related to POAG and positively correlated with visual field defects in POAG patients. Hence, ONOO- is a potential biomarker of POAG, especially advanced POAG. Besides, anti-nitration compounds may be novel ocular hypotensive agents based on the animal study.

Nitrodopamine modified MnO2 NS-MoS2QDs hybrid nanocomposite for the extracellular and intracellular detection of glutathione.

We have developed a highly sensitive and reliable fluorescence resonance energy transfer (FRET) probe using nitro-dopamine (ND) and dopamine (DA) coated MnO2 nanosheet (ND@MnO2 NS and DA@MnO2 NS) as an energy acceptor and MoS2 quantum dots (QDs) as an energy donor. By employing surface-modified MnO2 NS, we can effectively reduce the fluorescence intensity of MoS2 QDs through FRET. It can reduce MnO2 NS to Mn2+ and facilitate the fluorescence recovery of the MoS2 QDs. This ND@MnO2 NS@MoS2 QD-based nanoprobe demonstrates excellent sensitivity to GSH, achieving an LOD of 22.7 nM in an aqueous medium while exhibiting minimal cytotoxicity and good biocompatibility. Moreover, our sensing platform shows high selectivity to GSH towards various common biomolecules and electrolytes. Confocal fluorescence imaging revealed that the nanoprobe can image GSH in A549 cells. Interestingly, the ND@MnO2 NS nanoprobe demonstrates no cytotoxicity in living cancer cells, even at concentrations up to 100 μg mL-1. Moreover, the easy fabrication and eco-friendliness of ND@MnO2 NS make it a rapid and simple method for detecting GSH. We envision the developed nanoprobe as an incredible platform for real-time monitoring of GSH levels in both extracellular and intracellular mediums, proving valuable for biomedical research and clinical diagnostics.

A SLiM-dependent conformational change in baculovirus IE1 controls its focus formation ability.

The baculovirus IE1 gene encodes a multifunctional protein that is essential for both DNA replication and RNA transcription of the virus. Prior to viral DNA replication, IE1 promotes early gene transcription when localized in hr-dependent foci. During viral DNA replication, the IE1 foci expand and fuse to generate the virogenic stroma (VS) with IE1 found in the VS reticulum. To explore the IE1 structural features essential for this coordinated localization, we constructed various IE1 mutants based on three putative domains (N, I, and C). We determined that a BDI motif located in the intrinsic disorder region (IDR) between the N and I domains acts as a nuclear localization signal, whereas BDII and HLH in the C domain are required for VS localization in infected cells or for chromosomal association in uninfected mitotic cells. Deletion of the SLiM (short linear motif) located in the I domain restrains both nuclear- and VS localization. Intra-molecular fluorescence resonance energy transfer (FRET) probes of IE1 mutants revealed a conformational change of the I-C two-domain fragment during infection, which was inhibited by aphidicolin, suggesting that IE1 undergoes a stage-dependent conformational change. Further, homo-dimerization of the I domain and stage-dependent conformational changes require an intact SLiM. Mutational analysis of SLiM revealed that VS localization and chromosomal association were retained following S291A and S291E substitutions, but hr-dependent focus formation differed between the two mutations. These results suggest that coordinated IE1 localization is controlled by SLiM-dependent conformational changes that are potentially switched by the phosphorylation state of the SLiM.

Visualization of trans homophilic interaction of clustered protocadherin in neurons

Clustered protocadherin (Pcdh) functions as a cell recognition molecule through the homophilic interaction in the central nervous system. However, its interactions have not yet been visualized in neurons. We previously reported PcdhγB2-Förster resonance energy transfer (FRET) probes to be applicable only to cell lines. Herein, we designed γB2-FRET probes by fusing FRET donor and acceptor fluorescent proteins to a single γB2 molecule and succeeded in visualizing γB2 homophilic interaction in cultured hippocampal neurons. The γB2-FRET probe localized in the soma and neurites, and FRET signals, which were observed at contact sites between neurites, eliminated by ethylene glycol tetraacetic acid (EGTA) addition. Live imaging revealed that the FRET-negative γB2 signals rapidly moved along neurites and soma, whereas the FRET-positive signals remained in place. We observed that the γB2 proteins at synapses rarely interact homophilically. The γB2-FRET probe might allow us to elucidate the function of the homophilic interaction and the cell recognition mechanism.

A high-throughput screening platform for enzymes active on mucin-type O-glycoproteins.

Mucin-type O-glycosylation is a post-translational modification present at the interface between cells where it has important roles in cellular communication. However, deciphering the function of O-glycoproteins and O-glycans can be challenging, especially as few enzymes are available for their assembly or selective degradation. Here, to address this deficiency, we developed a genetically encoded screening methodology for the discovery and engineering of the diverse classes of enzymes that act on O-glycoproteins. The method uses Escherichia coli that have been engineered to produce an O-glycosylated fluorescence resonance energy transfer probe that can be used to screen for O-glycopeptidase activity. Subsequent cleavage of the substrate by O-glycopeptidases provides a read-out of the glycosylation state of the probe, allowing the method to also be used to assay glycosidases and glycosyltransferases. We further show the potential of this methodology in the first ultrahigh-throughput-directed evolution of an O-glycopeptidase.

Novel Förster Resonance Energy Transfer probe with quantum dot for a long-time imaging of active caspases inside individual cells

With the goal to investigate biological phenomena at a single-cell level, we designed, synthesized and tested a molecular probe based on Förster resonance energy transfer (FRET) between a highly luminescent quantum dot (QD) as a donor and a fluorophore or fluorescence quencher as an acceptor linked by a specific peptide. In principle, QD luminescence, effectively dissipated in the probe, is switched on after the cleavage of the peptide by a protease and the release of the quencher. We proposed a novel synthesis strategy of a probe. A two-step synthesis consists of: (i) Conjugation of CdTe QDs functionalized by –COOH groups of succinic acid on the nanoparticle surface with the designed specific peptide (GTADVEDTSC) using a ligand-exchange approach; (ii) A fast, high-yield reaction of amine-reactive succinimidyl group on the BHQ-2 quencher with N-terminal of the peptide. This way, any crosslinking between individual nanoparticles and any nonspecific conjugation bonds are excluded. The analysis of the product after the first step proved a high reaction yield and nearly no occurrence of unreacted QDs, a prerequisite of the specificity of our luminescent probe. Its parameters evaluated as Michaelis-Menten description of enzymatic kinetics are similar to products published by other groups. Our research is focused on the fluorescence microscopy analyses of biologically active molecules, such as proteolytic active caspases, playing important roles in cell signaling regulations in normal and diseased states. Consequently, they are attractive targets for clinical diagnosis and medical therapy. The ultimate goal of our work was to synthesize a new QD luminescent probe for a long-time quantitative monitoring of active caspase-3/7 distribution in apoptotic osteoblastic MC3T3-E1 cells treated with camptothecin. As a result of comparison, our synthetized luminescent probe provides longer imaging times of caspases than commercial products. The probe proved the stability of the luminescence signal inside cells for more than 14 days.

Genetically-encoded BRET probes enlight ligand bias-induced variable ion selectivity in TRPV1 and P2X5/7.

Whether ion channels experience ligand-dependent dynamic ion selectivity remains of critical importance since this could support ion channel functional bias. Tracking selective ion permeability through ion channels remains, however, challenging even with patch-clamp electrophysiology. In this study, we have developed highly-sensitive Bioluminescence Resonance Energy Transfer (BRET) probes providing dynamic measurements of Ca2+ and K+ concentrations and ionic strength in the nanoenvironment of TRPV1 and P2X channels pore in real-time and in live cells during drug challenges. Our results indicate that AMG517, BCTC, and AMG21629, three well-known TRPV1 inhibitors, more potently inhibit the CAPS-induced Ca2+ influx than the CAPS-induced K+ efflux through TRPV1. Even more strikingly, we found that AMG517, when injected alone, is a partial agonist of the K+ efflux through TRPV1 and triggers TRPV1-dependent cell membrane hyperpolarization. In a further effort to exemplify ligand bias in other families of cationic channels, using the same BRET-based strategy, we also detected concentration- and time-dependent ligand biases in P2X7 and P2X5 cationic selectivity when activated by Bz-ATP. These new custom-engineered BRET-based probes now open up avenues for adding value to ion-channel drug discovery platforms by taking ligand bias into account.

A Substituted-Rhodamine-Based Reversible Fluorescent Probe for In Vivo Quantification of Glutathione.

Quantifying glutathione (GSH) in cells and organisms is of great significance for understanding the mechanism of oxidative stress in various physiological and pathological processes. However, the quantification by fluorescence bioimaging in living tissues has much stricter requirements than the "Petri dish"-cultured cells in flat plates. Based on the evaluation of the electronic structure and steric hindrance-tuned reactivity of phospha-substituted rhodamine with GSH, a reversible Förster resonance energy transfer (FRET) probe ZpSiP with a distinct performance (Kd =4.9 mM, t1/2 =0.57 s, k=81 M-1 s-1 ) is developed for real time quantifying GSH in living cells. Furthermore, the near-infrared (NIR) probe succeeded in sensitively tracking the dynamics of GSH in the real organisms bearing tumors, chronic renal failure, and liver fibrosis for unveiling the related pathological processes. We believe that the advance in chemistry with quantitative analysis methods will initiate more promising progress and broad applications.

A Substituted‐Rhodamine‐Based Reversible Fluorescent Probe for In Vivo Quantification of Glutathione

AbstractQuantifying glutathione (GSH) in cells and organisms is of great significance for understanding the mechanism of oxidative stress in various physiological and pathological processes. However, the quantification by fluorescence bioimaging in living tissues has much stricter requirements than the “Petri dish”‐cultured cells in flat plates. Based on the evaluation of the electronic structure and steric hindrance‐tuned reactivity of phospha‐substituted rhodamine with GSH, a reversible Förster resonance energy transfer (FRET) probe ZpSiP with a distinct performance (Kd=4.9 mM, t1/2=0.57 s, k=81 M−1 s−1) is developed for real time quantifying GSH in living cells. Furthermore, the near‐infrared (NIR) probe succeeded in sensitively tracking the dynamics of GSH in the real organisms bearing tumors, chronic renal failure, and liver fibrosis for unveiling the related pathological processes. We believe that the advance in chemistry with quantitative analysis methods will initiate more promising progress and broad applications.

Theranostic FRET Gate to Visualize and Quantify Bacterial Membrane Breaching.

Designing new antimicrobial-cum-probes to study real-time bacterial membrane breaching and concurrently developing inquisitorial image-based analytical tools is essential for the treatment of infectious diseases. An array of aggregation-induced emission (AIE) polymers (donor) consisting of neutral, anionic, and cationic charges were designed and employed as antimicrobial theranostic gatekeepers for the permeabilization of the peptidoglycan layer-adherable crystal violet (CV, acceptor). An AIE-active tetraphenylethylene (TPE)-tagged polycaprolactone biodegradable platform was chosen, and their self-assembled tiny amphiphilic nanoparticles were employed as a gatekeeper in the construction of bacterial membrane-reinforced fluorescent resonance energy transfer (FRET) probes. Electrostatic adhering of the cationic AIE polymer and subsequent gate opening aided fluorescent FRET probe activation on the membrane of Gram-negative bacteria, Escherichia coli. The selective photoexcitation energy transfer process in confocal microscopy experiments facilitated the building of a visualization-based FRET assay for the quantification of bactericidal activity. Nonantimicrobial AIE polymers (neutral and anionic) did not breach the bacterial membrane, resulting in no FRET signal. Detailed photophysical studies were done to establish the FRET probe mechanism, and a proof of concept was established.

Genetic Encoding of a Fluorescent Noncanonical Amino Acid as a FRET Donor for theAnalysis of Deubiquitinase Activities.

The genetic code expansion technology enables the genetic encoding of fluorescent noncanonical amino acids (ncAAs) for site-specific fluorescent labeling of proteins. These co-translational and internal fluorescent tags have been harnessed to establish genetically encoded Förster resonance energy transfer (FRET) probes for studying protein structural changes and interactions. Here, we describe the protocols for site-specific incorporation of an aminocoumarin-derived fluorescent ncAA into proteins in E. coli and preparation of a fluorescent ncAA-based FRET probe for assaying theactivities of deubiquitinases, a key class of enzymes involved in ubiquitination. We also describe the deployment of an in vitro fluorescence assay to screen and analyze small-molecule inhibitors against deubiquitinases.

Outer Surface-Labeled Bacteria as Live Sensors Accurately Quantitating Interfacial pH: A Smart Technique for Antimicrobial Resistance.

The techniques to quantitatively monitor environmental factors surrounding the bacterial outer surface rather than the host's subcellular regions (e.g., lysosomes) should be the key to evaluate bacterial immune escape behavior. We report wild Staphylococcus aureus (SA) and methicillin-resistant Staphylococcus aureus (MRSA) labeled with a fluorescent resonance energy transfer probe, 4SR-L-BDP, on their outer surfaces as smart live sensors to quantify interfacial pH. The dual emission of 4SR-L-BDP affords high sensitivity to pH change in a ratiometric way in the pH range of 4-8 with high precision. Notably, 4SR-L-BDP possesses an anchoring group to fix on the bacterial surface for sensing the microenvironment encountered. Super-resolution imaging clearly demonstrates the specific labeling of bacterial membranes. These live sensors are applied in two-channel ratiometric imaging to dynamically visualize and quantify their interfacial pH changes during infection of macrophages. It is found that the interfacial pH of MRSA is lower by 0.2 units compared to that of SA. Such small but critical difference in pH reflects MRSA's ability to adapt to microenvironmental pH inside macrophages. These labeled bacteria as live sensors are also proven to be practically applicable in mice models with immune deficiency and immune activation.

Genetically-encoded BRET probes shed light on ligand bias–induced variable ion selectivity in TRPV1 and P2X5/7

Whether ion channels experience ligand-dependent dynamic ion selectivity remains of critical importance since this could support ion channel functional bias. Tracking selective ion permeability through ion channels, however, remains challenging even with patch-clamp electrophysiology. In this study, we have developed highly sensitive bioluminescence resonance energy transfer (BRET) probes providing dynamic measurements of Ca2+ and K+ concentrations and ionic strength in the nanoenvironment of Transient Receptor Potential Vanilloid-1 Channel (TRPV1) and P2X channel pores in real time and in live cells during drug challenges. Our results indicate that AMG517, BCTC, and AMG21629, three well-known TRPV1 inhibitors, more potently inhibit the capsaicin (CAPS)-induced Ca2+ influx than the CAPS-induced K+ efflux through TRPV1. Even more strikingly, we found that AMG517, when injected alone, is a partial agonist of the K+ efflux through TRPV1 and triggers TRPV1-dependent cell membrane hyperpolarization. In a further effort to exemplify ligand bias in other families of cationic channels, using the same BRET-based strategy, we also detected concentration- and time-dependent ligand biases in P2X7 and P2X5 cationic selectivity when activated by benzoyl-adenosine triphosphate (Bz-ATP). These custom-engineered BRET-based probes now open up avenues for adding value to ion-channel drug discovery platforms by taking ligand bias into account.

Genetic Analysis of the ATG16L1 c.898A>G (p.T300A) Variant in Acute and Chronic Pancreatitis.

Human and animal studies suggest an important role of autophagy in the pathogenesis of pancreatitis. ATG16L1 (autophagy-related 16 like 1) is part of a protein complex that is involved in the formation of autophagosomes. The c.898A > G (p.T300A) variant of ATG16L1 is associated with Crohn disease. In this study, we analyzed ATG16L1 c.898A > G (p.T300A) for an association with pancreatitis. We genotyped 777 patients and 551 control subjects of German origin by melting curve analysis using fluorescence resonance energy transfer probes. The patient group included 429 patients with nonalcoholic chronic pancreatitis (CP), 141 patients with alcoholic CP, and 207 patients with acute pancreatitis (AP). We classified AP by severity according to the Atlanta symposium 1992. Allele and genotype frequencies of ATG16L1 c.898A > G (p.T300A) did not differ significantly between patients and controls (G allele frequencies: nonalcoholic CP, 49.9%; alcoholic CP, 48.2%; AP, 49.5%; controls, 52.7%). We found no significant association with the severity of AP either. Our data do not support a role of ATG16L1 c.898A > G (p.T300A) in the pathogenesis of AP or CP or an influence on the severity of AP.

Generalized Bioluminescent Platform To Observe and Track Cellular Interactions.

Cell-to-cell communications are critical to biological processes ranging from embryonic development to cancer progression. Several imaging strategies have been developed to capture such interactions, but many are challenging to deploy in thick tissues and other complex environments. Here, we report a platform termed Luminescence to Observe and Track Intercellular Interactions (LOTIIS). The approach features split fragments of a luciferase enzyme that reassemble when target cells come into proximity. One fragment is secreted by "sender" cells, and the complementary piece is secreted by "receiver" cells. Split reporter assembly is facilitated by a single chain variable fragment (scFv)-peptide interaction on the receiver cell, resulting in localized light production. We demonstrate that LOTIIS can rapidly label cells in close proximity in a time- and distance-dependent fashion. The platform is also compatible with bioluminescence resonance energy transfer probes for multiplexed imaging. Collectively, these data suggest that LOTIIS will enable a variety of cellular interactions to be tracked in biological settings.

Synthesis and characterization of N-rich fluorescent bio-dots as a reporter in the design of dual-labeled FRET probe for TaqMan PCR: A feasibility study.

DNA-based analytical techniques have provided an advantageous sensing assay in the realm of biotechnology. Bio-inspired fluorescent nanodots are a novel type of biological staining agents with excellent optical properties widely used for cellular imaging and diagnostics. In the present research, we successfully synthesized bio-dots with excellent optical properties and high-quantum yield from DNA sodium salt through the hydrothermal method. We conjugated the bio-dots with 3' Eclipse Dark Quencher (Eclipse)-labeled single-strand oligodeoxyribonucleotide according to carbodiimide chemistry, to design a fluorescence resonance energy transfer (FRET) probe. The results confirmed the prosperous synthesis and surface functionalization of the bio-dot. Analysis of size, zeta potential, and FTIR spectroscopy verified successful bioconjugation of the bio-dots with probes. UV-visibility analysis and fluorescence intensity profile of the bio-dot and bio-dot@probes represented a concentration-dependent quenching of fluorescent signal of bio-dot by Eclipse after probe conjugation. The results demonstrated that TaqMan PCR was not feasible using the designed bio-dot@probes. Our results indicated that bio-dot can be used as an efficient fluorescent tag in the design of fluorescently labeled oligonucleotides with high biocompatibility and optical features.

An activatable fluorescence probe for visualization of DAGL activity in hippocampal tissue of brain-injured mice

Diacylglycerol lipase (DAGL) is an important biosynthetic enzyme of endocannabinoid 2-arachidonoyl-glycerol (2-AG) that activates the endocannabinoid receptors to mediate basic biological processes, such as inflammation, pain sensation, epileptogenesis, synaptic plasticity, neuroprotection and adult neurogenesis. However, the changes of DAGL activity are still unclear in neurophysiological and pathological processes due to a lack of effective method for in situ detection of DAGL activity. Herein, based on the specific cleavage of the sn-1 acyl group of diacylglycerol (DAG) hydrolyzed by DAGL, we rational design an activatable fluorescence probe by simulating the substrate of DAGL for detecting the DAGL activity. Coumarin and rhodamine fluorophores are introduced into 1- and 2-site of glycerol through ester bonds to construct a fluorescence resonance energy transfer probe CGR. The probe CGR shows high selectivity and can be used to detect the activity of endogenous DAGL in tissue and brain. Notably, we used the probe CGR to reveal the changes of DAGL activity after brain injury.

Improving Visualization of cAMP Gradients Using Algorithmic Modelling.

A ubiquitous second messenger molecule, cAMP is responsible for orchestrating many different cellular functions through a variety of pathways. Fӧrster resonance energy transfer (FRET) probes have been used to visualize cAMP spatial gradients in pulmonary microvascular endothelial cells (PMVECs). However, FRET probes have inherently low signal-to-noise ratios; multiple sources of noise can obscure accurate visualization of cAMP gradients using a hyperspectral imaging system. FRET probes have also been used to measure cAMP gradients in 3D; however, it can be difficult to differentiate between true FRET signals and noise. To further understand the effects of noise on experimental data, a model was developed to simulate cAMP gradients under experimental conditions. The model uses a theoretical cAMP heatmap generated using finite element analysis. This heatmap was converted to simulate the FRET probe signal that would be detected experimentally with a hyperspectral imaging system. The signal was mapped onto an image of unlabeled PMVECs. The result was a time lapse model of cAMP gradients obscured by autofluorescence, as visualized with FRET probes. Additionally, the model allowed the simulated expression level of FRET signal to be varied. This allowed accurate attribution of signal to FRET and autofluorescence. Comparing experimental data to the model results at different levels of FRET efficiency has allowed improved understanding of FRET signal specificity and how autofluorescence interferes with FRET signal detection. In conclusion, this model can more accurately determine cAMP gradients in PMVECs. This work was supported by NIH award P01HL066299, R01HL58506 and NSF award 1725937.