RESUMO
We introduce a general method to transform antibodies into ratiometric, bioluminescent sensor proteins for the no-wash quantification of analytes. Our approach is based on the genetic fusion of antibody fragments to NanoLuc luciferase and SNAP-tag, the latter being labeled with a synthetic fluorescent competitor of the antigen. Binding of the antigen, here synthetic drugs, by the sensor displaces the tethered fluorescent competitor from the antibody and disrupts bioluminescent resonance energy transfer (BRET) between the luciferase and fluorophore. The semisynthetic sensors display a tunable response range (submicromolar to submillimolar) and large dynamic range (ΔRmax >500 %), and they permit the quantification of analytes through spotting of the samples onto paper followed by analysis with a digital camera.
Assuntos
Anticorpos/química , Imunoensaio/métodos , Proteínas Luminescentes/química , Sistemas Automatizados de Assistência Junto ao Leito , Anticorpos/imunologia , Técnicas Biossensoriais , Regiões Determinantes de Complementaridade , Transferência de Energia , Humanos , Medições Luminescentes , Proteínas Luminescentes/imunologia , Metotrexato/química , Metotrexato/imunologia , Quinina/química , Quinina/imunologia , Reprodutibilidade dos Testes , Teofilina/química , Teofilina/imunologiaRESUMO
We introduce luciferases whose emission maxima can be tuned to different wavelengths by chemical labeling. The luciferases are chimeras of NanoLuc with either SNAP-tag or HaloTag7. Labeling of the self-labeling tag with a fluorophore shifts the emission maximum of NanoLuc to that of the fluorophore. Luciferases with tunable colors have applications as reporter genes, for the construction of biosensors and in bioimaging.
Assuntos
Luciferases/química , Técnicas Biossensoriais , Corantes Fluorescentes/química , Genes Reporter , Células HeLa , Humanos , Medições Luminescentes/métodosRESUMO
For many drugs, finding the balance between efficacy and toxicity requires monitoring their concentrations in the patient's blood. Quantifying drug levels at the bedside or at home would have advantages in terms of therapeutic outcome and convenience, but current techniques require the setting of a diagnostic laboratory. We have developed semisynthetic bioluminescent sensors that permit precise measurements of drug concentrations in patient samples by spotting minimal volumes on paper and recording the signal using a simple point-and-shoot camera. Our sensors have a modular design consisting of a protein-based and a synthetic part and can be engineered to selectively recognize a wide range of drugs, including immunosuppressants, antiepileptics, anticancer agents and antiarrhythmics. This low-cost point-of-care method could make therapies safer, increase the convenience of doctors and patients and make therapeutic drug monitoring available in regions with poor infrastructure.
Assuntos
Monitoramento de Medicamentos/métodos , Proteínas Luminescentes/química , Proteínas Recombinantes de Fusão/química , Antiarrítmicos/sangue , Anticonvulsivantes/sangue , Antineoplásicos/sangue , Técnicas Biossensoriais , Monitoramento de Medicamentos/economia , Monitoramento de Medicamentos/instrumentação , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Humanos , Processamento de Imagem Assistida por Computador , Imunossupressores/sangue , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Fotografação , Sistemas Automatizados de Assistência Junto ao Leito , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
We report the semisynthesis of a fluorescent glutamate sensor protein on cell surfaces. Sensor excitation at 547 nm yields a glutamate-dependent emission spectrum between 550 and 700 nm that can be exploited for ratiometric sensing. On cells, the sensor displays a ratiometric change of 1.56. The high sensitivity toward glutamate concentration changes of the sensor and its exclusive extracellular localization make it an attractive tool for glutamate sensing in neurobiology.
Assuntos
Técnicas Biossensoriais/métodos , Membrana Celular/química , Corantes Fluorescentes/química , Ácido Glutâmico/análise , Receptores de Glutamato/metabolismo , Membrana Celular/metabolismo , Expressão Gênica , Ácido Glutâmico/metabolismo , Células HEK293 , Humanos , Estrutura Terciária de Proteína , Receptores de Glutamato/química , Receptores de Glutamato/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Sensibilidade e EspecificidadeRESUMO
Progress in understanding signal transduction and metabolic pathways is hampered by a shortage of suitable sensors for tracking metabolites, second messengers, and neurotransmitters in living cells. Here we introduce a class of rationally designed semisynthetic fluorescent sensor proteins, called Snifits, for measuring metabolite concentrations on the cell surface of mammalian cells. Functional Snifits are assembled on living cells through two selective chemical labeling reactions of a genetically encoded protein scaffold. Our best Snifit displayed fluorescence intensity ratio changes on living cells significantly higher than any previously reported cell-surface-targeted fluorescent sensor protein. This work establishes a generally applicable and rational strategy for the generation of cell-surface-targeted fluorescent sensor proteins for metabolites of interest.
Assuntos
Técnicas Biossensoriais/métodos , Membrana Celular/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas Luminescentes/metabolismo , Animais , Células HEK293 , Humanos , Proteínas Luminescentes/genética , TransfecçãoRESUMO
The development of molecular probes to visualize cellular processes is an important challenge in chemical biology. One possibility to create such cellular indicators is based on the selective labeling of proteins with synthetic probes in living cells. Over the last years, our laboratory has developed different labeling approaches for monitoring protein activity and for localizing synthetic probes inside living cells. In this article, we review two of these labeling approaches, the SNAP-tag and CLIP-tag technologies, and their use for studying cellular processes.
Assuntos
Proteínas/metabolismo , Cálcio/metabolismo , Corantes Fluorescentes/metabolismoRESUMO
Monitoring metabolites at the point of care could improve the diagnosis and management of numerous diseases. Yet for most metabolites, such assays are not available. We introduce semisynthetic, light-emitting sensor proteins for use in paper-based metabolic assays. The metabolite is oxidized by nicotinamide adenine dinucleotide phosphate, and the sensor changes color in the presence of the reduced cofactor, enabling metabolite quantification with the use of a digital camera. The approach makes any metabolite that can be oxidized by the cofactor a candidate for quantitative point-of-care assays, as shown for phenylalanine, glucose, and glutamate. Phenylalanine blood levels of phenylketonuria patients were analyzed at the point of care within minutes with only 0.5 microliters of blood. Results were within 15% of those obtained with standard testing methods.
Assuntos
Técnicas de Transferência de Energia por Ressonância de Bioluminescência , Técnicas Biossensoriais , Proteínas de Escherichia coli/química , Monitorização Fisiológica/métodos , Testes Imediatos , Tetra-Hidrofolato Desidrogenase/química , Glicemia/análise , Proteínas de Escherichia coli/genética , Ácido Glutâmico/sangue , Humanos , NADP/metabolismo , Oxirredução , Fenilalanina/sangue , Fenilcetonúrias/sangue , Fenilcetonúrias/diagnóstico , Tetra-Hidrofolato Desidrogenase/genéticaRESUMO
Obtaining patient-specific information through the quantification of small molecules and proteins in bodily fluids is essential for personalized therapies. Point-of-care (POC) diagnostic devices hold the promise of delivering such benefit to a wide range of patients. However, there is a lack of enabling technology, as the majority of newly developed POC devices focus on the same underlying core technologies. Here we provide an overview of a new technology based on highly modular bioluminescent sensors that enables the quantification of small molecules and proteins at the POC with low-cost devices.
Assuntos
Técnicas Biossensoriais/métodos , Monitoramento de Medicamentos/métodos , Medições Luminescentes/métodos , Preparações Farmacêuticas/análise , Sistemas Automatizados de Assistência Junto ao Leito , Proteínas/análise , Animais , Técnicas Biossensoriais/instrumentação , Monitoramento de Medicamentos/instrumentação , Humanos , Medições Luminescentes/instrumentação , Modelos MolecularesRESUMO
Biosensors are used in many fields to measure the concentration of analytes, both in a cellular context and in human samples for medical care. Here, we outline the design of two types of modular biosensors: SNAP-tag-based indicators with a Fluorescent Intramolecular Tether (SNIFITs) and LUCiferase-based Indicators of Drugs (LUCIDs). These semisynthetic biosensors quantitatively measure analyte concentrations in vitro and on cell surfaces by an intramolecular competitive mechanism. We provide an overview of how to design and apply SNIFITs and LUCIDs.
Assuntos
Corantes Fluorescentes/química , Proteínas/genética , Técnicas Biossensoriais/métodos , Linhagem Celular , Escherichia coli/genética , Células HEK293 , Humanos , Engenharia de Proteínas/métodosRESUMO
We are introducing a new approach to evaluate cellular uptake of drugs and drug candidates into living cells. The approach is based on converting the protein target of a given class of compounds into a fluorescent biosensor. By measuring the binding of different compounds to their cognate biosensor in live cells and comparing these values to those measured in vitro, their cellular uptake and concentrations can be ranked. We demonstrate that our strategy enables the evaluation of the cellular uptake into the cytosol of 2 classes of inhibitors using two different sensor designs; first, sensors comprising the self-labeling protein SNAP conjugated with a chemically modified inhibitor shown for inhibitors of the enzyme human carbonic anhydrase II; and a label-free sensor for inhibitors of protein-protein interactions demonstrated for the protein pair p53-HDM2.
RESUMO
The possibility to design proteins whose activities can be switched on and off by unrelated effector molecules would enable applications in various research areas, ranging from biosensing to synthetic biology. We describe here a general method to modulate the activity of a protein in response to the concentration of a specific effector. The approach is based on synthetic ligands that possess two mutually exclusive binding sites, one for the protein of interest and one for the effector. Tethering such a ligand to the protein of interest results in an intramolecular ligand-protein interaction that can be disrupted through the presence of the effector. Specifically, we introduce a luciferase controlled by another protein, a human carbonic anhydrase whose activity can be controlled by proteins or small molecules in vitro and on living cells, and novel fluorescent and bioluminescent biosensors.
Assuntos
Técnicas Biossensoriais , Luciferases , Sítios de Ligação , Anidrases Carbônicas/metabolismo , Escherichia coli , Células HEK293 , Humanos , LigantesRESUMO
Nanoshuttles powered by the molecular motor kinesin have the potential to capture and concentrate rare molecules from solution as well as to transport, sort and assemble them in a high-throughput manner. One long-thought-of goal has been the realisation of a molecular assembly line with nanoshuttles as workhorses. To harness them for this purpose might allow the community to engineer novel materials and nanodevices. The central milestone towards this goal is to expose nanoshuttles to a series of different molecules or building blocks and load them sequentially to build hierarchical structures, macromolecules or materials. Here, we addressed this challenge by exploiting the synergy of two so far mostly complementary techniques, nanoshuttle-mediated active transport and pressure-driven passive transport, integrated into a single microfluidic device to demonstrate the realisation of a molecular assembly line. Multiple step protocols can thus be miniaturised to a highly parallelised and autonomous working lab-on-a-chip: in each reaction chamber, analytes or building blocks are captured from solution and are then transported by nanoshuttles across fluid flow boundaries in the next chamber. Cargo can thus be assembled, modified, analysed and eventually unloaded in a procedure that requires only one step by its operator.