A Reporter Cell Line for Screening Inhibitors of ADAR1
Tech Snapshot® captures today’s cutting-edge tools and technologies that will help drive drug discovery tomorrow. This installment was written by BPS Bioscience, a biotechnology development and manufacturing company focused on innovating proteins, antibodies, cell lines, lentiviruses and assay kits for biopharmaceutical and academic research.
BPS Bioscience strives to advance new scientific discoveries that lead to therapies by creating innovative start-to-finish solutions for research. We drive advancements in immunotherapy, epigenetics, coronavirus, cell signaling and other research areas, providing 4500+ high quality recombinant cell lines, virus-based tools, proteins and biochemical and cell-based assay kits focused on drug discovery. One of our core expertise areas is the engineering of cell lines for drug discovery, providing cell systems that enable high throughput compound screening using cellular readouts. Here, we describe our novel cell line for an emerging target in cancer and autoimmunity, ADAR1.
ADAR1 in Biology and Disease
Adenosine deaminases acting on RNA (ADAR) proteins catalyze the post-transcriptional modification of adenosine to inosine (A-to-I) on double-stranded RNA (dsRNA) molecules. ADARs edit A to I non-selectively in extended perfect double-stranded RNA duplexes, an activity that is of ancient origin and highly conserved. In humans, ADAR1 and ADAR2 have deaminating activity while ADAR3 lacks catalytic activity. ADAR1 has two isoforms, ADAR1p110 and ADAR1p150. ADAR1 functions as a major RNA editor, resulting in the differential regulation of RNA processing by other RNA-binding proteins.
Notably, ADAR1 regulates innate immunity by suppressing pattern recognition receptor mechanisms. During viral infection, long stretches of exogenous dsRNA are recognized by MDA5, PKR, and OAS, initiating three pathways leading to apoptosis to prevent viral spread. The MDA5 pathway leads to induction of interferon (IFN) genes, which are critical in fighting viral infections. In the absence of viral infection, endogenous sequences of dsRNA are modified by ADAR1 preventing their recognition by these innate immune sensors. Although essential in cellular discrimination of exogenous or endogenous dsRNA, modifications of RNA by ADAR1 can directly lead to amino acid substitutions, which can lead to disease. Furthermore, ADAR1 also contributes to alterations in the processing and function of micro (miRNAs) both positively and negatively.
Importantly, ADAR1 overactivity or loss of function have both been linked to various cancers and to how cancers can potentially be treated. With the emergence of checkpoint blockade immunotherapy, researchers continue to search for mechanisms of enhancing efficacy, especially against solid tumors. ADAR1, itself, may function as an immune checkpoint. Studies have now identified that inactivating ADAR1 in tumor cells renders them highly susceptible to immunotherapy, a process linked to unedited dsRNA and activation of pattern recognition receptors inducing IFN pathways.
ADAR1 Dual Luciferase Reporter HEK293 Cell Line
Based on a similar reporter system described by K. Fritzell, et al. 2019. Nucleic Acids Res. 47(4):e22, BPS Bioscience has released the first commercially available luciferase-based reporter cell line for screening inhibitors of ADAR1 (Catalog #78547). This cell line expresses ADAR1 under the control of a CMV promoter and an ADAR-editing RNA reporter construct expressed under the control of a separate CMV promoter. The reporter contains the gene encoding Firefly luciferase, which is constitutively expressed in the cells, upstream of the gene encoding the GluA2 ADAR substrate followed by the Renilla luciferase gene. The sequence corresponding to GluA2 has been modified to contain an amber stop codon (UAG). When edited by ADAR this stop codon (UAG) will be changed to code for tryptophan (UIG, where the I is interpreted translationally as a G), resulting in the expression of Renilla luciferase (A). Conversely, in the absence of ADAR1 activity, translation terminates at the stop codon and Renilla is not expressed. Reporter activity is read out as Renilla luciferase/Firefly luciferase ratio, whereby upon inhibition of ADAR activity (B), the UAG (stop) codon is read, resulting in a decrease in expression of Renilla luciferase and a decrease in the Renilla luciferase/Firefly luciferase ratio.
This cell system serves as an excellent solution for high throughput compound screening to identify specific ADAR1 inhibitors. Shown below: Dose response of ADAR1 Dual Luciferase Reporter HEK293 cells to Fludarabin-Cl. The cells were treated with increasing doses of Fludarabin-Cl for 30 hours and both Firefly and Renilla luciferase activity were measured using Dual Luciferase (Firefly-Renilla) Assay System. Results are shown as the Renilla/Firefly luciferase ratio.
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- B. Song, et al. 2022. Wiley Interdiscip Rev RNA. 13(10:e1665.
- L. Xu and M. Ohman. 2018. Genes (Basel). 10(1):12.
- M. Lamers, et al. 2019. Front Immunol. 10:1763.
- K. Fritzell, et al. 2019. Nucleic Acids Res. 47(4):e22.