Modulating protein activity using tethered ligands with mutually exclusive binding sites

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

A Semisynthetic Fluorescent Sensor Protein for Glutamate

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

Bioluminescent sensor proteins for point-of-care therapeutic drug monitoring

To ensure efficacy but avoid toxicity, many drugs require monitoring of their concentrations in the patient’s blood during therapy. Currently, therapeutic drug monitoring (TDM) is done in diagnostic laboratories that need trained personnel and expensive instruments. For this reason, the technique is only used when absolutely necessary and patients in regions with poor infrastructure cannot benefit from it at all. Even though methods that can be used at bedside or at home would have obvious advantages in terms of therapeutic outcome and convenience, none exist yet. This thesis introduces a new class of bioluminescent sensor proteins that could fill this need. LUCIDs (LUCiferase-based Indicators for Drugs) rely on the blue emission of a luciferase and the analyte-dependent resonance energy transfer to an orange fluorophore. LUCIDs provide a strong signal and an exceptional dynamic range, permitting measurements by spotting minimal sample volumes on paper and recording emission using a simple digital camera. We present the construction of such sensor proteins for the anticancer agent methotrexate, the antiepileptic topiramate, the immunosuppressants tacrolimus, sirolimus, and cyclosporin A, as well as the cardiac glycoside digoxin. Finally, we show that LUCIDs can precisely quantify drug concentrations in samples from hospital patients. 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

Means and methods for bioluminescence resonance energy transfer (bret) analysis in a biological sample

The invention relates to the field of in vitro detection methods using luminescence. Provided is a sensor molecule for detecting an analyte of interest in a sample using bioluminescence resonance energy transfer (BRET), the sensor molecule comprising a proteinaceous moiety tethered to a synthetic regulatory molecule. Also provided is an analytical device comprising a sensor and methods using the sensor molecule

Sensor molecules and uses thereof

The invention generally relates to methods, reagents and devices for determining a concentration of an analyte in a sample. Provided is a proteinaceous sensor molecule for detecting an analyte of interest (A), comprising a signal generating moiety tethered to a synthetic regulatory molecule capable of modulating signal generation by intramolecular binding, wherein (i) the synthetic regulatory molecule comprises a primary ligand (L1) capable of intramolecular binding to a primary partner (BP1) on the sensor molecule; wherein (ii) the sensor molecule comprises a secondary ligand (L2) capable of binding to a secondary binding partner (BP2) which is A or a binding partner thereof; and wherein (iii) binding of L1 and L2 to their respective binding partners is mutually exclusive, such that binding of BP2 to L2 influences binding of L1 to BP1, resulting in a conformational change within the sensor molecule such that the generated signal is modulated

Rational Design and Applications of Semisynthetic Modular Biosensors: SNIFITs and LUCIDs

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

Highly Modular Bioluminescent Sensors for Small Molecules and Proteins

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