In vitro selection of specific RNA aptamers for the NFAT DNA binding domain.
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.
Cho
Jung-Sun
JS
Department of Molecular Biology, Institute of Nanosensor and Biotechnology, Dankook University, Seoul 140-714, Korea.
Lee
Young Ju
YJ
Shin
Kyung-Sook
KS
Jeong
Sujin
S
Park
Jungchan
J
Lee
Seong-Wook
SW
D000595:Amino Acid Sequence; D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; D012313:RNA