NanoBRET™ Assays for Monitoring Protein Interactions in Living Cells
Poster Oct 13, 2017
Jacqui L. Méndez, Kristin Riching, Sarah Mahan, Nancy Murphy, Marie Schwinn, Thomas Machleidt, Keith Wood, Marjeta Urh, and Danette L. Daniels
Identifying small molecule modulators, inhibitors or activators, of protein-protein interactions (PPI) remains challenging, largely due to the difficulty of developing robust, high-throughput screening tools that can used to interrogate these interactions within a biologically relevant context. Bioluminescent resonance energy transfer (BRET) has been used to monitor real-time protein:protein interactions in live cells, but current approaches suffer from limited sensitivity and narrow dynamic range. Here we present a new BRET method, termed NanoBRET, based on a small and extremely bright NanoLuc luciferase coupled to a HaloTag - long-wavelength fluorophore. This highly effective energy donor-acceptor combination boosts BRET performance, and the higher sensitivity facilitates application of the method in high density plates and high throughput screening. Here we present several examples of protein interaction biology that can be interrogated with NanoBRET including epigenetic interactions, adaptor recruitment to membrane receptors, and proteasomal recruitment of proteins targeted for degradation.
We utilized paired synthetic crRNAs coupled with our synthetic tracrRNA in cells transduced with lentiviral Cas9 to perform a functional knockout on hsa-miR-221. This three-part system (crRNA, tracrRNA and Cas9) has demonstrated efficient gene editing when used with only one guide RNA, but the goal was to use two crRNAs to remove the entire stem-loop.READ MORE