Genetically Encoded FRET-Based Nanosensor for Real-Time Monitoring of A549 Exosomes: Early Diagnosis of Cancer.

Exosomes contain a plethora of unique disease biomarkers involving cellular homeostasis, infection dissemination, cancer development, and cardiac diseases. Exosomes originating from cancer cells have promising biomarkers for the early detection and assessment of the therapeutic response to cancer. The exosomal epidermal growth factor receptor (EGFR) is a potential biomarker which is overexpressed in cancer; thus, the level of EGFR expression is investigated by so many methods in a liquid and solid biopsy. The optimal method for isolating pure exosomal EGFRs has not been well understood so far. Current approaches are complicated and time-consuming, therefore hampering their clinical applications. Here, we demonstrate the creation of an innovative fluorescence resonance energy transfer (FRET) sensor, named ExoSen (exosome sensor), which can be implemented to determine the concentration of exosomal EGFRs at in vitro as well as in vivo levels. In this study, a sensing element for A549 exosomes, mitogen-inducible gene 6 (MIG6), has been employed between the FRET pair ECFP and Venus. MIG6 binding to ExoSen induced a conformational change that can be monitored by a variation in the FRET ratio. Moreover, the developed sensor, expressed in bacterial, yeast, and HEK-293T cells, demonstrates an increased FRET ratio with the addition of A549 exosomes, which can quantify the A549 exosomes noninvasively. The ExoSen enables rapid detection of A549 exosomes with great sensitivity at a concentration of 3.5 × 109 particles/mL. ExoSen is stable to pH fluctuations and provides a highly accurate, real-time optical readout in cell-based experiments by using confocal microscopy.

FRET-based probe for ratiometric detection and imaging of folic acid in real-time.

Inadequate folic acid intake is linked to diseases such as megaloblastic anemia, neural tube defects, and hyperhomocysteinemia, increasing the risk of vascular disease and thrombosis. Folic acid, a cofactor in various enzymes, can be produced by plants and bacteria, but not by humans and other animals. L-5-methyl-tetrahydrofolate (L-5-methyl-THF) is the primary dietary folate form, transported in circulation for cellular metabolism. Traditional methods of determining folic acid levels are unreliable and time-consuming. SenFol (Sensor for folic acid) is a fluorescence resonance energy transfer (FRET)-based nanosensor that we have developed by inserting folic acid-binding protein (FolT) as the folate detecting domain between the pair of enhanced cyan fluorescent protein (ECFP) and Venus. The developed sensor is highly specific, produces a quick signal, which is pH stable, and delivers precise, ratiometric readings in cell-based experiments. The projected affinity score of folic acid with FolT was -7.4 kcal/mol. The apparent affinity (Kd) of SenFol for folic acid is 28.49 × 10-9 M, with a detection range of 5 × 10-9 M to 5 × 10-7 M, and a maximum FRET ratio change of 0.45. WT SenFol, a highly efficient folic acid nanosensor, can dynamically detect intracellular folic acid content in E. coli, yeast, and HEK-293 T cells, confirming its potential.

FRET-based nanosensors for monitoring and quantification of alcohols in living cells.

Odorants constitute a small and chemically diverse group of molecules with ethanol functioning as a key odorant that induces reproductive toxicity and adverse chronic effects on the liver. Analytical tools designed so far for the detection of odorant molecules are relatively invasive. Therefore, a tool that can measure the corresponding rate changes of ethanol concentration in real-time is highly desirable. Here in this work, we report a genetically encoded fluorescence resonance energy transfer (FRET)-based nanosensor for in vivo quantification of ethanol at the cellular level with high spatial and temporal resolution. A human odorant-binding protein (hOBPIIa) was flanked by fluorescent proteins ECFP (Enhanced Cyan Fluorescent Protein) and Venus at the N- and C-terminus respectively. The constructed FRET nanosensor was named the fluorescent indicator protein for odorants (FLIPO). FLIPO allows in vitro and in vivo determination of FRET changes in a concentration-dependent manner. The developed nanosensor is highly specific to ethanol, stable to pH changes and provides rapid detection rate response. FLIPO-42 is the most efficient nanosensor created that measures ethanol with an apparent affinity (Kd) of 4.16 µM and covers the physiological range of 500 nM to 12 µM ethanol measurement. FLIPO-42 can measure ethanol dynamics in bacterial, yeast and mammalian cells non-invasively in real time which proves its efficacy as a sensing device in both prokaryotic and eukaryotic systems. Taken together, a prototype for a set of nanosensors was established, potentially enabling the monitoring of dynamic changes of ethanol and investigate its uptake and metabolism with subcellular resolution in vivo and ex vivo. Furthermore, the advent of a set of novel nanosensors will provide us with the tools for numerous medical, scientific, industrial and environmental applications which would help to illuminate their role in biological systems.

Genetically encoded FRET-based optical sensor for Hg2+ detection and intracellular imaging in living cells.

Due to the potential toxicity of mercury, there is an immediate need to understand its uptake, transport and flux within living cells. Conventional techniques used to analyze Hg2+ are invasive, involve high cost and are less sensitive. In the present study, a highly efficient genetically encoded mercury FRET sensor (MerFS) was developed to measure the cellular dynamics of Hg2+ at trace level in real time. To construct MerFS, the periplasmic mercury-binding protein MerP was sandwiched between enhanced cyan fluorescent protein (ECFP) and venus. MerFS is pH stable, offers a measurable fluorescent signal and binds to Hg2+ with high sensitivity and selectivity. Mutant MerFS-51 binds with an apparent affinity (Kd) of 5.09 × 10-7 M, thus providing a detection range for Hg2+ quantification between 0.210 µM and 1.196 µM. Furthermore, MerFS-51 was targeted to Escherichia coli (E. coli), yeast and human embryonic kidney (HEK)-293T cells that allowed dynamic measurement of intracellular Hg2+ concentration with a highly responsive saturation curve, proving its potential application in cellular systems.

Nanoscale gizmos - the novel fluorescent probes for monitoring protein activity.

Nanobiotechnology has emerged inherently as an interdisciplinary field, with collaborations from researchers belonging to diverse backgrounds like molecular biology, materials science and organic chemistry. Till the current times, researchers have been able to design numerous types of nanoscale fluorescent tool kits for monitoring protein-protein interactions through real time cellular imagery in a fluorescence microscope. It is apparent that supplementing any protein of interest with a fluorescence habit traces its function and regulation within a cell. Our review therefore highlights the application of several fluorescent probes such as molecular organic dyes, quantum dots (QD) and fluorescent proteins (FPs) to determine activity state, expression and localization of proteins in live and fixed cells. The focus is on Fluorescence Resonance Energy Transfer (FRET) based nanosensors that have been developed by researchers to visualize and monitor protein dynamics and quantify metabolites of diverse nature. FRET based toolkits permit the resolution of ambiguities that arise due to the rotation of sensor molecules and flexibility of the probe. Achievements of live cell imaging and efficient spatiotemporal resolution however have been possible only with the advent of fluorescence microscopic technology, equipped with precisely sensitive automated softwares.

Leishmania donovani modulates host miRNAs regulating cholesterol biosynthesis for its survival.

Cholesterol reduction by intracellular protozoan parasite Leishmania donovani (L. donovani), causative agent of leishmaniasis, impairs antigen presentation, pro-inflammatory cytokine secretion and host-protective membrane-receptor signaling in macrophages. Here, we studied the miRNA mediated regulation of cholesterol biosynthetic genes to understand the possible mechanism of L. donovani-induced cholesterol reduction and therapeutic importance of miRNAs in leishmaniasis. System-scale genome-wide microtranscriptome screening was performed to identify the miRNAs involved in the regulation of expression of key cholesterol biosynthesis regulatory genes through miRanda3.0. 11 miRNAs out of 2823, showing complementarity with cholesterol biosynthetic genes were finally selected for expression analysis. These selected miRNAs were differentially regulated in THP-1 derived macrophages and in primary human macrophages by L. donovani. Correlation of expression and target validation through luciferase assay suggested two key miRNAs, hsa-miR-1303 and hsa-miR-874-3p regulating the key genes hmgcr and hmgcs1 respectively. Inhibition of hsa-mir-1303 and hsa-miR-874-3p augmented the expression of targets and reduced the parasitemia in macrophages. This study will also provide the platform for the development of miRNA-based therapy against leishmaniasis.

FRET-Based Genetically Encoded Sensor to Monitor Silver Ions.

Silver is commonly used in wound dressing, photography, health care products, laboratories, pharmacy, biomedical devices, and several industrial purposes. Silver (Ag+) ions are more toxic pollutants widely scattered in the open environment by natural processes and dispersed in soil, air, and water bodies. Ag+ binds with metallothionein, macroglobulins, and albumins, which may lead to the alteration of various enzymatic metabolic pathways. To analyze the uptake and metabolism of silver ions in vitro as well as in cells, a range of high-affinity fluorescence-based nanosensors has been constructed using a periplasmic protein CusF, a part of the CusCFBA efflux complex, which is involved in providing resistance against copper and silver ions in Escherichia coli. This nanosensor was constructed by combining of two fluorescent proteins (donor and acceptor) at the N- and C-terminus of the silver-binding protein (CusF), respectively. SenSil (WT) with a binding constant (K d) of 5.171 µM was more efficient than its mutant variants (H36D and F71W). This nanosensor allows monitoring the level of silver ions in real time in prokaryotes and eukaryotes without any disruption of cells or tissues.

Enhanced sensitivity and detection range of FRET-based vitamin B12 nanosensor.

Vitamin B12 (cobalamin) is a cobalt-containing compound that acts as an essential co-factor for various enzymes involved in the metabolic processes of the living cells. The constructed FRET Sensor for Vitamin Anemia Linked (SenVitAL) displayed marginal FRET efficiency. Here, we report the development of a molecular SenVitAL containing enhanced cyan fluorescent protein (ECFP) and venus as FRET pair to improve the FRET efficiency for optical imaging and screening of already developed sensor by our group. The sensor is the improved version of previously reported SenVitAL and consists of ECFP/venus as FRET pair instead of the originally used pair CFP/YFP. To increase the physiological range of vitamin B12 measurement, affinity mutants were created. Compared to the wild type, SenVitAL-5 with W44Q mutation has higher affinity and displayed large dynamic detection range (0.10-480 µM) in response to vitamin B12 binding. For cell-based monitoring and dynamic measurement of vitamin B12 flux rates, SenVitAL-5 was successfully expressed in cytosol of yeast and mammalian cells. Changes in the emission intensities of the two fluorophores were detected using confocal microscopy in both cell types in response to vitamin B12. With the addition of 50 µM extracellular vitamin B12 to the cells, the emission intensity of venus increased and that of ECFP decreased over the time. Furthermore, the results show that the variant SenVitAL-5 measures the vitamin B12 in a concentration-dependent manner, showing the resulting increase in the FRET ratio and thus confirming its utility as an ideal fluorescent indicator for the detection of vitamin B12 in eukaryotic systems in real time.

A Fluorescence Resonance Energy Transfer-Based Analytical Tool for Nitrate Quantification in Living Cells.

Nitrate (NO3 -) is a critical source of nitrogen (N) available to microorganisms and plants. Nitrate sensing activates signaling pathways in the plant system that impinges upon, developmental, molecular, metabolic, and physiological responses locally, and globally. To sustain, the high crop productivity and high nutritional value along with the sustainable environment, the study of rate-controlling steps of a metabolic network of N assimilation through fluxomics becomes an attractive strategy. To monitor the flux of nitrate, we developed a non-invasive genetically encoded fluorescence resonance energy transfer (FRET)-based tool named "FLIP-NT" that monitors the real-time uptake of nitrate in the living cells. The developed nanosensor is suitable for real-time monitoring of nitrate flux in living cells at subcellular compartments with high spatio-temporal resolution. The developed FLIP-NT nanosensor was not affected by the pH change and have specificity for nitrate with an affinity constant (K d) of ∼5 µM. A series of affinity mutants have also been generated to expand the physiological detection range of the sensor protein with varying K d values. It has been found that this sensor successfully detects the dynamics of nitrate fluctuations in bacteria and yeast, without the disruption of cellular organization. This FLIP-NT nanosensor could be a very important tool that will help us to advance the understanding of nitrate signaling.

Real time quantification of intracellular nickel using genetically encoded FRET-based nanosensor.

Nickel (Ni2+) is an essential mineral nutrient that is required in trace quantities, but exposure to excess amounts can be toxic or carcinogenic. Mechanisms underlying Ni2+ imbalance and toxicity are poorly understood because most analytical methods currently used to probe this metal are invasive or also have toxic effects. To address these problems, a genetically encoded FRET-based probe for nickel metal (FProNiM) was constructed for real-time detection of Ni2+ in live cells. FProNiM was constructed by sandwiching the bacterial periplasmic nickel-binding protein NikA between the fluorescent protein variants ECFP and Venus. Ni2+ binding to FProNiM induced a conformational change that could be detected by a change in fluorescence resonance energy transfer (FRET) ratio (acceptor/donor fluorescence). In vitro studies demonstrated that FProNiM is specific for Ni2+, insensitive to changes in pH from 5.0 to 8.0, and has an affinity (Kd) of 21.6 µM, which allows detection of Ni2+ concentrations from 0.1 to 5000 µM. The sensor variant FProNiM-5 was also expressed in Escherichia coli (E. coli), yeast, and mammalian cells. In each cell type, changes in Ni2+ concentrations were detected with subcellular resolution using confocal microscopy.

Engineering genetically encoded FRET-based nanosensors for real time display of arsenic (As3+) dynamics in living cells.

Arsenic poisoning has been a major concern that causes severe toxicological damages. Therefore, intricate and inclusive understanding of arsenic flux rates is required to ascertain the cellular concentration and establish the carcinogenetic mechanism of this toxicant at real time. The lack of sufficiently sensitive sensing systems has hampered research in this area. In this study, we constructed a fluorescent resonance energy transfer (FRET)-based nanosensor, named SenALiB (Sensor for Arsenic Linked Blackfoot disease) which contains a metalloregulatory arsenic-binding protein (ArsR) as the As3+ sensing element inserted between the FRET pair enhanced cyan fluorescent protein (ECFP) and Venus. SenALiB takes advantage of the ratiometic FRET readout which measures arsenic with high specificity and selectivity. SenALiB offers rapid detection response, is stable to pH changes and provides highly accurate, real-time optical readout in cell-based assays. SenALiB-676n with a binding constant (Kd) of 0.676 × 10-6 M is the most efficient affinity mutant and can be a versatile tool for dynamic measurement of arsenic concentration in both prokaryotes and eukaryotes in vivo in a non-invasive manner.

Role of green fluorescent proteins and their variants in development of FRET-based sensors.

Since the last decade, a lot of advancement has been made to understand biological processes involving complex intracellular pathways. The major challenge faced was monitoring and trafficking of metabolites in real time. Although a range of quantitative and imaging techniques have been developed so far, the discovery of green fluorescent proteins (GFPs) has revolutionized the advancement in the field of metabolomics. GFPs and their variants have enabled researchers to 'paint' a wide range of biological molecules. Fluorescence resonance energy transfer (FRET)-based genetically encoded sensors is a promising technology to decipher the real-time monitoring of the cellular events inside living cells. GFPs and their variants, due to their intrinsic fluorescence properties, are extensively being used nowadays in cell-based assays. This review focuses on structure and function of GFP and its derivatives, mechanism emission and their use in the development of FRET-based sensors for metabolites.