RESUMEN
Heterotrimeric G-proteins (Gαßγ) are the main transducers of signals from GPCRs, mediating the action of countless natural stimuli and therapeutic agents. However, there are currently no robust approaches to directly measure the activity of endogenous G-proteins in cells. Here, we describe a suite of optical biosensors that detect endogenous active G-proteins with sub-second resolution in live cells. Using a modular design principle, we developed genetically encoded, unimolecular biosensors for endogenous Gα-GTP and free Gßγ: the two active species of heterotrimeric G-proteins. This design was leveraged to generate biosensors with specificity for different heterotrimeric G-proteins or for other G-proteins, such as Rho GTPases. Versatility was further validated by implementing the biosensors in multiple contexts, from characterizing cancer-associated G-protein mutants to neurotransmitter signaling in primary neurons. Overall, the versatile biosensor design introduced here enables studying the activity of endogenous G-proteins in live cells with high fidelity, temporal resolution, and convenience.
Asunto(s)
Transferencia de Energía por Resonancia de Bioluminiscencia/instrumentación , Transferencia de Energía por Resonancia de Bioluminiscencia/métodos , Técnicas Biosensibles/instrumentación , Técnicas Biosensibles/métodos , Guanosina Trifosfato/metabolismo , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Secuencias de Aminoácidos , Animales , Células Cultivadas , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/química , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Factores de Intercambio de Guanina Nucleótido/antagonistas & inhibidores , Guanosina Trifosfato/química , Células HEK293 , Células HeLa , Humanos , Ratones , Ratones Endogámicos C57BL , Mutación , Neoplasias/genética , Neoplasias/metabolismo , Neuronas/química , Neuronas/metabolismo , Neuronas/fisiología , Transducción de Señal , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/metabolismoRESUMEN
In recent years, considerable efforts have been done to better understand the peculiar emission properties of upconverting materials due to their widespread applications in different and important technological fields such as upconversion-based photoactivated cancer therapies, photoactivated drug-delivery, magnetic resonance imaging contrast agents, bioimaging. However, one of the most promising applications of upconverting materials concerns the field of sensing, due to their unique emission properties. In fact, the minimal autofluorescence, blinking, photo-bleaching, and high photostability makes them an excellent alternative to organic dyes or quantum dots. This article reviews the state-of-the-art, design, and sensing strategies of upconversion-based sensing platforms, with special attention to upconverting nanoparticles, as well as how the incorporation of these materials into pre-existing diagnostic tests and bioassays have improved their capabilities for the detection of different kinds of analytes.
Asunto(s)
Bioensayo , Transferencia de Energía por Resonancia de Bioluminiscencia/métodos , Técnicas Biosensibles/instrumentación , Medios de Contraste/síntesis química , Sistemas de Liberación de Medicamentos , Sondas Moleculares/síntesis química , Transferencia de Energía por Resonancia de Bioluminiscencia/instrumentación , Cromatografía de Afinidad/instrumentación , Cromatografía de Afinidad/métodos , Humanos , Imagen por Resonancia Magnética/instrumentación , Imagen por Resonancia Magnética/métodos , Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Analíticas Microfluídicas/métodos , Nanopartículas/química , Nanopartículas/metabolismo , Imagen Óptica/instrumentación , Imagen Óptica/métodos , Procesos FotoquímicosRESUMEN
IL-1ß processing is one of the hallmarks of inflammasome activation and drives the initiation of the inflammatory response. For decades, Western blot or ELISA have been extensively used to study this inflammatory event. Here, we describe the use of a bioluminescence resonance energy transfer (BRET) biosensor to monitor IL-1ß processing in real time and in living macrophages either using a plate reader or a microscope.
Asunto(s)
Transferencia de Energía por Resonancia de Bioluminiscencia/instrumentación , Técnicas Biosensibles/métodos , Interleucina-1beta/metabolismo , Animales , Línea Celular , Humanos , Inflamasomas/metabolismo , Macrófagos/citología , Macrófagos/inmunología , RatonesRESUMEN
Due to the complicated media, monitoring proteases in real physiological environments is still a big challenge. Bioluminescence resonance energy transfer (BRET) is one of the promising techniques but its application is limited by the susceptibility to buffer composition, which might cause serious errors for the assay. Herein we report a novel combination of BRET pair with humanized Gaussia luciferase (hGluc) and highly bright red fluorescence protein tdTomato for sensitive and robust protease activity determination. As a result, the hGluc/tdTomato BRET pair showed much better tolerance to buffer composition, pH and sample matrices, and wide spectral separation (Δλ:~110 nm). With the protease sensor built with this pair, the detection limit for enterokinase reached 2.1 pM in pure buffer and 3.3 pM in 3% serum. The proposed pair would find broad use in both in vitro and in vivo assays, especially for samples with complicated matrix.
Asunto(s)
Transferencia de Energía por Resonancia de Bioluminiscencia/métodos , Técnicas Biosensibles/métodos , Luciferasas/química , Proteínas Luminiscentes/química , Péptido Hidrolasas/metabolismo , Proteínas Recombinantes de Fusión/química , Animales , Transferencia de Energía por Resonancia de Bioluminiscencia/instrumentación , Técnicas Biosensibles/instrumentación , Enteropeptidasa/sangre , Límite de Detección , Luciferasas/genética , Proteínas Luminiscentes/genética , Plásmidos , Conejos , Proteínas Recombinantes de Fusión/genética , Soluciones , Proteína Fluorescente RojaRESUMEN
Here we report the design of a bioluminescence resonance energy transfer (BRET)-based sensing system that could detect nucleic acid target in 5 min with high sensitivity and selectivity. The sensing system is based on adjacent binding of oligonucleotide probes labeled with Renilla luciferase (Rluc) and quantum dot (Qd) on the nucleic acid target. Here Rluc, a bioluminescent protein that generates light by a chemical reaction, is employed as an energy donor, and a quantum dot is used as an energy acceptor. Bioluminescence emission of Rluc overlaps with the Qd absorption whereas the emission of Qd is shifted from the emission of Rluc allowing for monitoring of BRET. In the presence of target, the labeled probes bind adjacently in a head-to-head fashion leading to BRET from Rluc to Qd upon addition of a substrate coelenterazine. The sensing system could detect target nucleic acid in buffer as well as in Escherichia coli cellular matrix in 5 min with a detection limit of 0.54 pmol. The ability to detect target nucleic acid rapidly in a cellular matrix with high sensitivity will prove highly beneficial in biomedical and environmental applications.
