Activity-responsive Split RNA Polymerase Platform for Biosensors and Gene Therapy
SUMMARY
- An understanding of protein-protein interactions (PPIs) is critical to uncovering new disease pathophysiology and for developing small molecule and antibody treatments. Problematically, current approaches to assay PPIs are limited by sensitivity, signal to noise ratio, and the need to carefully optimize the assay for each new application.
- The inventors engineered a new class of PPI sensor, activity-responsive split RNA polymerase (AR), which is a split RNA polymerase/protein fusion. When the fused proteins interact in close proximity, the RNA polymerase fully assembles to produce a unique transcriptional event. This approach allows PPIs to be indirectly quantified with proteomics or genomics.
- The invention is a biosensing platform for monitoring specific PPIs in vivo. The platform can be programmed to monitor therapeutic efficacy in real-time, or for event-specific gene therapy applications.
- An in vitro proof-of-concept study was conducted in which the responsiveness of HEK293T cells to BCL-2 and MCL-1 (cancer small molecule PPI inhibitors) was assayed with the AR system. The level of PPIs was assayed through GFP and RFP expression.
FIGURE
Spit RNAP biosensors can simultaneously monitor pharmacological engagement of two protein-protein interactions in live cells. HEK293T cells were treated with ABT-119 (Venetoclax, green) and/or a preclinical small molecule, A121 (red), and inhibitory activity was measured by AR system (decreased fluorescence signal).
ADVANTAGES
ADVANTAGES
- Improved signal to noise ratio and sensitivity
- Multiplexable
- Highly programmable (input and response)
APPLICATIONS
- Diagnostics
- Therapeutics
- Biosensors
- Gene Therapy
- Pre-clinical drug monitoring
PUBLICATIONS
- Pu, J; et al. RNA polymerase tags to monitor multidimensional protein-protein interactions reveal pharmacological engagement of Bcl-2 protiens. J AM Chem Soc. 2017 Aug 30; 139(34):11964-11972.
- Pu, J; Kentala, K; Dickinson, BC. Multidimensional control of Cas9 by evolved RNA polymerase-based biosensors. ACS Chem Biol. 2018 Feb 16; 13(2): 431-437.
- Pu, J; et al. Evolution of a C-terminal modification tolerance in full-length and split T7 RNA polymerase biosensors. Chembiochem. 2019 Jun 14;20(12):1547-1553.
- US:16/305,298