4 research outputs found
Chemically induced dimerization modules as a platform for plant biosensor engineering
Protein biosensors for small molecules have important applications in agriculture, medicine, and security, but it remains difficult to rapidly produce a high-affinity sensor for a given ligand. This is partly due to two major challenges. First, most small molecule ligands have only a small number of residues with which a protein can make energetically favorable contacts, making it difficult to engineer high-affinity binding. Second, even if a high-affinity binding protein is engineered, it is difficult to transduce the binding event into an output. The majority of plant hormone perception occurs by chemically induced dimerization, where binding of the hormone to a soluble receptor causes a conformational change that allows the receptor to form a heterodimer with an interaction partner. These CID modules make an ideal platform for engineering small molecule biosensors because they naturally address the two primary challenges above: their unique architecture allows sensitive biosensors to be constructed from low-affinity receptors and protein dimerization provides a natural method of ligand binding transduction. The ability to engineer CID modules would lead directly to in planta biosensors and would also have broader applications to biosensor design in other biological systems. Here we describe the development of a general biosensor engineering platform using the abscisic acid receptor PYR1 of Arabidopsis thaliana, which was previously engineered to sense the agrochemical mandipropamid.1 We combine comprehensive mutagenesis2,3, high-throughput screening, deep sequencing, and machine learning to rapidly construct a model of the fitness landscape for binding of PYR1 to a specific ligand. We then use this model to design a targeted library to screen for higher affinity sensors. For high-throughput screening, we use both an established yeast two-hybrid (Y2H) screen and a novel yeast surface display (YSD) system. These techniques offer complementary advantages: Y2H is straightforward to implement and requires no purified protein, while YSD offers higher throughput and more stringent quantification of protein-protein interactions. Finally, we describe early development of two additional CID modules from the gibberellin and strigolactone sensing networks of A. thaliana. (1) Park, S.-Y.; Peterson, F. C.; Mosquna, A.; Yao, J.; Volkman, B. F.; Cutler, S. R. Agrochemical Control of Plant Water Use Using Engineered Abscisic Acid Receptors. Nature 2015, 520 (7548), 545–548. https://doi.org/10.1038/nature14123. (2) Wrenbeck, E. E.; Klesmith, J. R.; Stapleton, J. A.; Adeniran, A.; Tyo, K. E. J.; Whitehead, T. A. Plasmid-Based One-Pot Saturation Mutagenesis. Nat. Methods 2016, 13 (11), 928–930. https://doi.org/10.1038/nmeth.4029. (3) Medina-Cucurella, A. V.; Steiner, P. J.; Faber, M. S.; Beltrán, J.; Borelli, A. N.; Kirby, M. B.; Cutler, S. R.; Whitehead, T. A. User-Defined Single Pot Mutagenesis Using Unpurified Oligo Pools. Re
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High-Performance Cannabinoid Sensor Empowered by Plant Hormone Receptors and Antifouling Magnetic Nanorods.
The misuse of cannabinoids and their synthetic variants poses significant threats to public health, necessitating the development of advanced techniques for detection of these compounds in biological or environmental samples. Existing methods face challenges like lengthy sample pretreatment and laborious antifouling steps. Herein, we present a novel sensing platform using magnetic nanorods coated with zwitterionic polymers for the simple, rapid, and sensitive detection of cannabinoids in biofluids. Our technique utilizes the engineered derivatives of the plant hormone receptor Pyrabactin Resistance 1 (PYR1) as drug recognition elements and employs the chemical-induced dimerization (CID) mechanism for signal development. Additionally, the magnetic nanorods facilitate efficient target capture and reduce the assay duration. Moreover, the zwitterionic polymer coating exhibits excellent antifouling capability, preserving excellent sensor performance in complex biofluids. Our sensors detect cannabinoids in undiluted biofluids like serum, saliva, and urine with a low limit of detection (0.002 pM in saliva and few pM in urine and serum) and dynamic ranges spanning up to 9 orders of magnitude. Moreover, the PYR1 derivatives demonstrate high specificity even in the presence of multiple interfering compounds. This work opens new opportunities for sensor development, showcasing the excellent performance of antifouling magnetic nanorods that can be compatible with different recognition units, including receptors and antibodies, for detecting a variety of targets
Rapid biosensor development using plant hormone receptors as reprogrammable scaffolds
A general method to generate biosensors for user-defined molecules could provide detection tools for a wide range of biological applications. Here, we describe an approach for the rapid engineering of biosensors using PYR1 (Pyrabactin Resistance 1), a plant abscisic acid (ABA) receptor with a malleable ligand-binding pocket and a requirement for ligand-induced heterodimerization, which facilitates the construction of sense-response functions. We applied this platform to evolve 21 sensors with nanomolar to micromolar sensitivities for a range of small molecules, including structurally diverse natural and synthetic cannabinoids and several organophosphates. X-ray crystallography analysis revealed the mechanistic basis for new ligand recognition by an evolved cannabinoid receptor. We demonstrate that PYR1-derived receptors are readily ported to various ligand-responsive outputs, including enzyme-linked immunosorbent assay (ELISA)-like assays, luminescence by protein-fragment complementation and transcriptional circuits, all with picomolar to nanomolar sensitivity. PYR1 provides a scaffold for rapidly evolving new biosensors for diverse sense-response applications