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Fluorogenic biosensors are essential tools widely used in biomedicine, chemical biology, environmental protection and food safety. Fluorescence resonance energy transfer (FRET) is a crucial technique for developing fluorogenic biosensors that provide mechanistic insight into bioprocesses through time‐spatial bioimaging in living cells and organisms. Although extensive FRET‐based sensors have been developed for detecting or imaging analytes of interest over the past decade, few comprehensive reviews have summarized the recent studies from the fundamental chemical angle about the design and application. In this work, the recent advance in the discovery of FRET biosensors using donor‐acceptor dye combinations is described and they are classified based on different types of analytes, such as mall molecules, proteins, enzymes, nucleic acids and metal ions. This review provides molecular‐level inspiration for the design of FRET‐based biosensors, aiding in their application in biosensing and bioimaging. The construction of the FRET donor‐acceptor pairs and the application in biosensing of analytes of interest including proteins, ions, nucleic acids and small molecules.

Granzyme B (GZMB) is a key enzyme released by cytotoxic T lymphocytes (CTL) and natural killer (NK) cells to induce apoptosis in target cells. We designed a novel fluorogenic biosensor which is able to assess GZMB activity in a specific and sensitive manner. This cleavage-responsive sensor for T cell activity level (CRSTAL) is based on a fluorescent protein that is only activated upon cleavage by GZMB or caspase-8. CRSTAL was tested in stable cell lines and demonstrated a strong and long-lasting fluorescence signal upon induction with GZMB. It can detect GZMB activity not only by overexpression of GZMB in target cells but also following transfer of GZMB and perforin from effector cells during cytotoxicity. This feature has significant implications for cancer immunotherapy, particularly in monitoring the efficacy of chimeric antigen receptor (CAR)-T cells. CAR-T cells are a promising therapy option for various cancer types, but monitoring their activity in vivo is challenging. The development of biosensors like CRSTAL provides a valuable tool for monitoring of CAR-T cell activity. In summary, CRSTAL is a highly sensitive biosensor that can detect GZMB activity in target cells, providing a means for evaluating the cytotoxic activity of immune cells and monitoring T cell activity in real time.

Small molecules are highly relevant targets for detection and quantification. They are also used to diagnose and monitor the progression of disease and infectious processes and track the presence of contaminants. Fluorogenic RNA-based biosensors (FRBs) represent an appealing solution to the problem of detecting these targets. They combine the portability of molecular systems with the sensitivity and multiplexing capacity of fluorescence, as well as the exquisite ligand selectivity of RNA aptamers. In this review, we first present the different sensing and reporting aptamer modules currently available to design an FRB, together with the main methodologies used to discover modules with new specificities. We next introduce and discuss how both modules can be functionally connected prior to exploring the main applications for which FRB have been used. Finally, we conclude by discussing how using alternative nucleotide chemistries may improve FRB properties and further widen their application scope.

We developed a fluorogenic RNA aptamer-based burden biosensor in Escherichia coli capable of informing on the burden of different genetic constructs via dynamic fluorescence readout.We characterised a library of Pepper and Broccoli aptamers and highlighted that aptamer relative fluorescence strength is dependent on promoter, host and growth context.We revealed that aptamer expression impacts Escherichia coli, leading to decrease growth in an RNA-scaffold-dependent manner, calling for caution in adopting these aptamers for applications in live cells.We tested a library of burden biosensors and showed the tRNA-Broc biosensor capable of reporting on the burden imposed by different expression levels and different genetic constructs.The biosensor developed here quickly responded to burden and enabled identification of best-performing genetic variants with reduced burden providing a tool for improved engineering in Escherichia coli. Cell burden impacts the performance of engineered genetic constructs with great interest towards the development of tools to track it and improve biotechnology applications. Fluorogenic RNA aptamers are excellent candidates for live monitoring of burden because their production is expected to impose negligible load on the host. Here, we characterised a library of aptamers when expressed from different promoters in two Escherichia coli strains. We found that aptamer performance is dependent on the context of expression, and that, contrary to expectation, aptamer production impacts host fitness. We then built a library of burden-responsive biosensors testing their response to heterologous expression. The tRNA-Broc biosensor was selected for its fluorescence response, minimal impact on growth and ability to differentiate the burden imposed by different expression levels and constructs. The biosensor developed here adds to the collection of tools available to characterise burden and support applications where improved host performance is sought. [Display omitted] The intracellular fluorescent aptamer-based burden biosensor reported here is a novel tool available to characterise the level of cellular burden imposed by heterologous genetic constructs in Escherichia coli. This tool has been characterised with behavioural observations and proof of concept validation in the laboratory, corresponding to a technology readiness level (TRL) of 3 or 4. Aptamer-based biosensors of burden are now ready for large scale validation and test in industrial settings. Indeed, a crucial next step in the development of this tool is to measure burden imposed by larger libraries of synthetic constructs, as well as validating its use in bioreactors. While this study highlights an unexpected burden imposed by fluorogenic aptamers on the bacterial hosts, such burden is negligeable when these aptamers are expressed from the endogenous promoter htpG1 thus not representing a limitation in the adoption of the biosensor for burden screening. Better understanding of the type of burden sensed by these biosensors will improve their application. Fluorogenic RNA aptamers are adopted to design a burden-responsive biosensor in Escherichia coli. While these aptamers can impact bacterial growth, the tRNA-Broc biosensor enables sensing of the burden imposed by different genetic constructs and identification of low-burden variants with improved performance.

Our ability to observe biochemical events with high spatial and temporal resolution is essential for understanding the functioning of living systems. Intrinsically fluorescent proteins such as the green fluorescent protein (GFP) have revolutionized the way biologists study cells and organisms. The fluorescence toolbox has been recently extended with new fluorescent reporters composed of a genetically encoded tag that binds endogenously present or exogenously applied fluorogenic chromophores (so-called fluorogens) and activates their fluorescence. This review presents the toolbox of fluorogen-based reporters and biosensors available to biologists. Various applications are detailed to illustrate the possible uses and opportunities offered by this new generation of fluorescent probes and sensors for advanced bioimaging.

Oxidative stress has been causally linked to various diseases. Electron transport chain (ETC) inhibitors such as rotenone and antimycin A are frequently used in model systems to study oxidative stress. Oxidative stress that is provoked by ETC inhibitors can be visualized using the fluorogenic probe 2′,7′-dichlorodihydrofluorescein-diacetate (DCFH2-DA). Non-fluorescent DCFH2-DA crosses the plasma membrane, is deacetylated to 2′,7′-dichlorodihydrofluorescein (DCFH2) by esterases, and is oxidized to its fluorescent form 2′,7′-dichlorofluorescein (DCF) by intracellular ROS. DCF fluorescence can, therefore, be used as a semi-quantitative measure of general oxidative stress. However, the use of DCFH2-DA is complicated by various protocol-related factors that mediate DCFH2-to-DCF conversion independently of the degree of oxidative stress. This study therefore analyzed the influence of ancillary factors on DCF formation in the context of ETC inhibitors. It was found that ETC inhibitors trigger DCF formation in cell-free experiments when they are co-dissolved with DCFH2-DA. Moreover, the extent of DCF formation depended on the type of culture medium that was used, the pH of the assay system, the presence of fetal calf serum, and the final DCFH2-DA solvent concentration. Conclusively, experiments with DCFH2-DA should not discount the influence of protocol-related factors such as medium and mitochondrial inhibitors (and possibly other compounds) on the DCFH2-DA-DCF reaction and proper controls should always be built into the assay protocol.

Aptasensors became popular instruments in bioanalytical chemistry and molecular biology. To increase specificity, perspective signaling elements in aptasensors can be separated into a G-quadruplex (G4) part and a free fluorescent dye that lights up upon binding to the G4 part. However, current systems are limited by relatively low enhancement of fluorescence upon dye binding. Here, we added duplex modules to G4 structures, which supposedly cause the formation of a dye-binding cavity between two modules. Screening of multiple synthetic GFP chromophore analogues and variation of the duplex module resulted in the selection of dyes that light up after complex formation with two-module structures and their RNA analogues by up to 20 times compared to parent G4s. We demonstrated that the short duplex part in TBA25 is preferable for fluorescence light up in comparison to parent TBA15 molecule as well as TBA31 and TBA63 stabilized by longer duplexes. Duplex part of TBA25 may be partially unfolded and has reduced rigidity, which might facilitate optimal dye positioning in the joint between G4 and the duplex. We demonstrated dye enhancement after binding to modified TBA, LTR-III, and Tel23a G4 structures and propose that such architecture of short duplex-G4 signaling elements will enforce the development of improved aptasensors.

Lactate is a key metabolite with applications ranging from monitoring training efficiency to early sepsis detection and monitoring the metabolic state of cell cultures. In this study, a paper-based lactate sensor utilizing a fluorescent readout was developed. Unlike common lactate dehydrogenase (LDH)-based methods, these sensors use a green fluorescent protein (GFP) or mApple-coupled lactate binding domain, which provides a fluorescent readout upon lactate binding. We demonstrate that immobilizing these proteins on paper does not affect their ability to bind lactate and produce a fluorescent readout, by monitoring lactate levels in the cell culture supernatant applying different cell culture conditions.

Genetically encodable sensors have been widely used in the detection of intracellular molecules ranging from metal ions and metabolites to nucleic acids and proteins. These biosensors are capable of monitoring in real-time the cellular levels, locations, and cell-to-cell variations of the target compounds in living systems. Traditionally, the majority of these sensors have been developed based on fluorescent proteins. As an exciting alternative, genetically encoded RNA-based molecular sensors (GERMS) have emerged over the past few years for the intracellular imaging and detection of various biological targets. In view of their ability for the general detection of a wide range of target analytes, and the modular and simple design principle, GERMS are becoming a popular choice for intracellular analysis. In this review, we summarize different design principles of GERMS based on various RNA recognition modules, transducer modules, and reporting systems. Some recent advances in the application of GERMS for intracellular imaging are also discussed. With further improvement in biostability, sensitivity, and robustness, GERMS can potentially be widely used in cell biology and biotechnology.

S. cerevisiae RNR3 and RAD54 gene transcription becomes strongly activated upon DNA damage. This property was used to construct yeast strains in which DNA damage can be monitored by a very sensitive fluorogenic assay in a convenient 96-well microtiter plate format. These strains carried stably integrated fusions of RNR3 or RAD54 promoters to the E. coli beta-glucuronidase GUS gene. GUS activity was measured by fluorogenic detection, a method that greatly increases the precision and sensitivity of the assay. Detection levels were similar to those of real-time quantitative PCR methods and close to the limits of biological response. The two reporters differed in terms of fold-induction, activation kinetics, sensitivity and specificity upon exposure to a variety of genotoxic compounds. While RNR3-GUS showed the fastest response, RAD54-GUS showed the highest sensitivity: similar to previous reported sensitivities for bacterial and eukaryotic genotoxic detection systems. These reporter strains may complement current genotoxicity tests, but they also have the advantages of higher flexibility, requirement for shorter incubation times, and the capability of being fully automated. In addition, the intrinsic features of the system facilitate its easy improvement by genetic manipulating the yeast strain or by introducing mammalian metabolizing enzymes.