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In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing
Published on: May 5, 2023
In vitro selection of specific RNA inhibitors of NFATc
Soo-Jin Bae1, Ji-Hyun Oum, Surendra Sharma
1Department of Molecular Biology, Dankook University, San 8, Hannam-Dong, Yongsan-Gu, Seoul 140-714, Republic of Korea.
Abstract:
NFAT (nuclear factor of activated T cells) plays a pivotal role in inducible gene transcription during the immune response and functions as a major target for immunosuppressive drugs such as cyclosporin A and FK-506. However, due to toxic effects of these drugs, which arise from their ability to inhibit calcineurin in non-immune cells, development of agents that directly target NFAT without toxic effects is warranted. Here, we present an in vitro selection of RNA aptamer to NFATc DNA binding domain (DBD) from a combinatorial RNA library with 41 nucleotide-long random sequences using the SELEX technique. The selected (SE) RNA was found to specifically and avidly bind NFATc DBD based on immunoprecipitation and competitive gel retardation assay. SE RNA also efficiently and specifically inhibited DNA binding capacity of NFATc, but not NFATp. Furthermore, transient RNA transfection studies show that only SE RNA can selectively and efficiently inhibit the NFATc- but neither the NFkappaB- nor NFATp-driven promoter activity in cells. These results suggest that SE RNA identified in this study is a specific inhibitor of NFATc activation, and hence, can be used not only for the study of NFAT functions but for the development of potent immune modulating agents.
Insights
Researchers developed a novel RNA aptamer that specifically targets and inhibits NFATc, a key protein in immune responses. This discovery offers a potential alternative to current immunosuppressants, minimizing toxic side effects for immune modulation research.
Area of Science:
- Molecular Biology
- Immunology
- Drug Discovery
Background:
- Nuclear factor of activated T cells (NFAT) is crucial for immune response gene transcription.
- Current immunosuppressants targeting NFAT, like cyclosporin A, have toxic side effects due to calcineurin inhibition in non-immune cells.
Purpose of the Study:
- To develop novel agents that directly target NFAT without causing systemic toxicity.
- To identify and characterize a specific RNA aptamer inhibitor of NFATc.
Main Methods:
- Systematic Evolution of Ligands by Exponential Enrichment (SELEX) to select RNA aptamers against NFATc DNA binding domain (DBD).
- Immunoprecipitation and competitive gel retardation assays to confirm binding specificity and affinity.
- Transient RNA transfection assays to assess inhibition of NFATc-driven promoter activity in cells.
Main Results:
- A specific RNA aptamer (SE RNA) was selected that avidly binds the NFATc DBD.
- SE RNA demonstrated specific inhibition of NFATc DNA binding and NFATc-driven promoter activity, but not NFATp or NFkappaB.
- The aptamer showed selective inhibition of NFATc activation in cellular assays.
Conclusions:
- The identified SE RNA is a specific inhibitor of NFATc activation.
- This SE RNA can be utilized for studying NFAT functions and developing new immune-modulating agents with reduced toxicity.
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