Related Experiment Video
Updated: Aug 22, 2026
An Optimized Quantitative Pull-Down Analysis of RNA-Binding Proteins Using Short Biotinylated RNA
Published on: February 17, 2023
In vitro selection of specific RNA aptamers for the NFAT DNA binding domain
Jung-Sun Cho1, Young Ju Lee, Kyung-Sook Shin
1Department of Molecular Biology, Institute of Nanosensor and Biotechnology, Dankook University, Seoul 140-714, Korea.
Abstract:
Nuclear factor of activated T cells (NFAT) plays a central role in the immune response, and the immuno-suppressive drugs, cyclosporin A and FK-506, have been developed to inhibit it. However, due to the toxic effects of these drugs, which derive from their ability to inhibit calcineurin in non-immune tissues, the identification of small compounds that target NFAT directly could be an approach to developing less toxic immunosuppressive therapy. Using an in vitro selection technology termed SELEX on a combinatorial RNA library with 40 nucleotide-long random sequences, we have isolated two RNA aptamers to the NFAT DNA binding domain (DBD). Gel retardation assays and surface plasmon resonance measurements showed that the aptamers have a specific and high affinity (apparent KD~10 to 100 nM) for the NFAT DBD. Enzymatic probing analysis showed that the two RNA aptamers have similar structures and share a sequence that forms an apical loop. Moreover, RNase footprinting analysis showed that the shared sequence (GATATGAAGGA/ TGTG/AGAGAG) is critical for binding to both NFATp DBD and NFATc DBD. These results suggest that short RNAs identified in this study is a specific aptamer to NFAT DBD, and hence could be applied not only for the delineation of NFAT functions but for the development of potent immune modulating lead compounds.
Insights
Researchers developed novel RNA aptamers targeting the Nuclear Factor of activated T cells (NFAT) DNA binding domain. These aptamers offer a potential pathway for less toxic immunosuppressive therapies by directly inhibiting NFAT.
Area of Science:
- Immunology
- Molecular Biology
- Drug Discovery
Background:
- Nuclear factor of activated T cells (NFAT) is crucial in immune responses.
- Current immunosuppressants like cyclosporin A and FK-506 inhibit NFAT but cause toxicity by affecting calcineurin in non-immune tissues.
Purpose of the Study:
- To identify small compounds that directly target NFAT.
- To develop a less toxic immunosuppressive therapy approach.
Main Methods:
- Utilized in vitro SELEX technology on a combinatorial RNA library.
- Isolated two RNA aptamers specific to the NFAT DNA binding domain (DBD).
- Employed gel retardation assays, surface plasmon resonance, enzymatic probing, and RNase footprinting to characterize aptamer binding and structure.
Main Results:
- Two high-affinity RNA aptamers (apparent KD ~10-100 nM) were isolated for the NFAT DBD.
- A shared apical loop sequence in the aptamers was identified as critical for binding to both NFATp DBD and NFATc DBD.
- The identified RNA aptamers demonstrate specific binding to NFAT DBD.
Conclusions:
- The study successfully identified specific RNA aptamers targeting the NFAT DBD.
- These aptamers hold potential for elucidating NFAT functions.
- They represent promising lead compounds for developing novel, potent immune-modulating therapies with reduced toxicity.
