TY - JOUR
T1 - Antagonistic RNA aptamer specific to a heterodimeric form of human interleukin-17A/F
AU - Adachi, Hironori
AU - Ishiguro, Akira
AU - Hamada, Michiaki
AU - Sakota, Eri
AU - Asai, Kiyoshi
AU - Nakamura, Yoshikazu
N1 - Funding Information:
We thank E. Inomata and K. Ito for technical instructions and discussion, and C. Crist for his help in preparing the manuscript. This work was supported in part by a Core Research for Evolution Science and Technology (CREST) grant from the Japan Science and Technology Agency, and research grants from The Ministry of Education, Sports, Culture, Science and Technology of Japan (MEXT) and from Ministry of Health, Labour and Welfare .
PY - 2011/7
Y1 - 2011/7
N2 - Interleukin-17 (IL-17) is a pro-inflammatory cytokine produced primarily by a subset of CD4+ T cells, called Th17 cells, that is involved in host defense, inflammation and autoimmune disorders. The two most structurally related IL-17 family members, IL-17A and IL-17F, form homodimeric (IL-17A/A, IL-17F/F) and heterodimeric (IL-17A/F) complexes. Although the biological significance of IL-17A and IL-17F have been investigated using respective antibodies or gene knockout mice, the functional study of IL-17A/F heterodimeric form has been hampered by the lack of an inhibitory tool specific to IL-17A/F. In this study, we aimed to develop an RNA aptamer that specifically inhibits IL-17A/F. Aptamers are short single-stranded nucleic acid sequences that are selected in vitro based on their high affinity to a target molecule. One selected aptamer against human IL-17A/F, AptAF42, was isolated by repeated cycles of selection and counterselection against heterodimeric and homodimeric complexes, respectively. Thus, AptAF42 bound IL-17A/F but not IL-17A/A or IL-17F/F. The optimized derivative, AptAF42dope1, blocked the binding of IL-17A/F, but not of IL-17A/A or IL-17F/F, to the IL-17 receptor in the surface plasmon resonance assay in vitro. Consistently, AptAF42dope1 blocked cytokine GRO-α production induced by IL-17A/F, but not by IL-17A/A or IL-17F/F, in human cells. An RNA footprinting assay using ribonucleases against AptAF42dope1 in the presence or absence of IL-17A/F revealed that part of the predicted secondary structure fluctuates between alternate forms and that AptAF42dope1 is globally protected from ribonuclease cleavage by IL-17A/F. These results suggest that the selected aptamer recognizes a global conformation specified by the heterodimeric surface of IL-17A/F.
AB - Interleukin-17 (IL-17) is a pro-inflammatory cytokine produced primarily by a subset of CD4+ T cells, called Th17 cells, that is involved in host defense, inflammation and autoimmune disorders. The two most structurally related IL-17 family members, IL-17A and IL-17F, form homodimeric (IL-17A/A, IL-17F/F) and heterodimeric (IL-17A/F) complexes. Although the biological significance of IL-17A and IL-17F have been investigated using respective antibodies or gene knockout mice, the functional study of IL-17A/F heterodimeric form has been hampered by the lack of an inhibitory tool specific to IL-17A/F. In this study, we aimed to develop an RNA aptamer that specifically inhibits IL-17A/F. Aptamers are short single-stranded nucleic acid sequences that are selected in vitro based on their high affinity to a target molecule. One selected aptamer against human IL-17A/F, AptAF42, was isolated by repeated cycles of selection and counterselection against heterodimeric and homodimeric complexes, respectively. Thus, AptAF42 bound IL-17A/F but not IL-17A/A or IL-17F/F. The optimized derivative, AptAF42dope1, blocked the binding of IL-17A/F, but not of IL-17A/A or IL-17F/F, to the IL-17 receptor in the surface plasmon resonance assay in vitro. Consistently, AptAF42dope1 blocked cytokine GRO-α production induced by IL-17A/F, but not by IL-17A/A or IL-17F/F, in human cells. An RNA footprinting assay using ribonucleases against AptAF42dope1 in the presence or absence of IL-17A/F revealed that part of the predicted secondary structure fluctuates between alternate forms and that AptAF42dope1 is globally protected from ribonuclease cleavage by IL-17A/F. These results suggest that the selected aptamer recognizes a global conformation specified by the heterodimeric surface of IL-17A/F.
KW - Cytokine GRO-α
KW - IL-17A/F heterodimer
KW - Induction
KW - Interleukin-17
KW - RNA aptamer
KW - RNA footprint
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U2 - 10.1016/j.biochi.2011.04.003
DO - 10.1016/j.biochi.2011.04.003
M3 - Article
C2 - 21524680
AN - SCOPUS:79958026711
VL - 93
SP - 1081
EP - 1088
JO - Biochimie
JF - Biochimie
SN - 0300-9084
IS - 7
ER -