In vitro selection of specific RNA inhibitors of NFATc.
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.
Bae
Soo-Jin
SJ
Department of Molecular Biology, Dankook University, San 8, Hannam-Dong, Yongsan-Gu, Seoul 140-714, Republic of Korea.
Oum
Ji-Hyun
JH
Sharma
Surendra
S
Park
Jungchan
J
Lee
Seong-Wook
SW
D001483:Base Sequence; D020650:Combinatorial Chemistry Techniques; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D011485:Protein Binding; D012313:RNA / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D014157:Transcription Factors / Q000037:antagonists & inhibitors* / Q000378:metabolism
D004268:DNA-Binding Proteins; D050778:NFATC Transcription Factors; C494003:NFATC1 protein, human; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; D012313:RNA