The technology uses Receptor Compartmentalized Partnered Replication (RCPR) to identify optimal interactions between receptors or pathways and their effector molecules by selectively amplifying genes that produce a thermostable polymerase during thermal cycling in emulsified libraries.
Background
The technology involves the identification and evolution of receptor and signal transduction pathway interactions with effector molecules. This is crucial for advancing our understanding of cellular communication and for the development of targeted therapies.
Traditional methods of studying these interactions often involve labor-intensive and low-throughput techniques, which are not efficient for large-scale applications. They also often require growth assays that can confound signaling with fitness effects, leading to the selection of "cheaters" that improve growth through mutations outside the target gene. Additionally, these methods lack negative selection mechanisms to eliminate native or off-target ligand interactions, necessitating extensive screening processes. There is the difficulty of identifying compounds that are truly specific to their target receptors without affecting other neural receptors and interactions in the brain, as well.
No existing technology allows for the simultaneous production of a compound in an organism and the functional modulation of a signal transduction pathway, nor does it conveniently enable barcoding and amplification based on receptor activation.
Technology description
The technology described involves methods and platforms utilizing Receptor Compartmentalized Partnered Replication (CPR) to identify associations between signal transduction pathways or receptors and their effector molecules. This system links a partner gene, which can be a receptor, signal transduction pathway, or metabolic pathway, to the production of a thermostable polymerase. Libraries of organisms are emulsified with primers that amplify the partner gene. During thermal cycling of the emulsion, genes that efficiently produce the thermostable polymerase are selectively amplified. This process allows for the identification of the most effective partner genes in producing the polymerase, thereby facilitating the selection and evolution of receptors or pathways that interact optimally with specific effector molecules.
The technology is differentiated by its ability to perform high-throughput screening and directed evolution of receptors and pathways in a single, integrated process. Unlike traditional methods that may require separate steps for screening and evolution, CPR combines these into a streamlined workflow. This is achieved by linking the production of a thermostable polymerase to the function of the partner gene, allowing for direct selection based on polymerase production. The use of emulsification ensures that each library member is compartmentalized, preventing crosstalk and enabling precise selection. Additionally, the technology supports the co-identification of metabolic pathways and their interacting receptors, making it a powerful tool for drug discovery, receptor modification, and the development of orthogonal receptor-effector pairs.
Benefits
- Facilitates identification of associations between signal transduction pathways (or receptors) and their effector molecules
- Enables selective amplification of genes that efficiently produce thermostable polymerase
- Allows for the selection and evolution of receptors or pathways that optimally interact with specific effector molecules
- Provides a method for high-throughput screening of receptors
- Can be used to evolve receptors responsive to orthogonal ligands
- Useful for the selection of metabolic pathways or pathway variants that produce compounds activating a particular receptor
- Enables co-identification of metabolic pathways and receptors that functionally interact
- Allows for drug discovery and screening of off-target drug effects
- Provides a novel negative selection method for easy validation of orthogonal receptor-ligand pairs
Commercial applications
- Drug discovery
- Receptor modification
- Pathway modification
- Metabolic pathway selection
- Therapeutic antibody selections
Patent
Issued patent US 11,746,374
