Antagonistic RNA aptamer specific to a heterodimeric form of human interleukin-17A/F
Antagonistic RNA aptamer specific to a heterodimeric form of human interleukin-17A/F
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DOI：
10.1016/j.biochi.2011.04.003
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发表时间：
2011-07-01
期刊：
影响因子：
3.9
通讯作者：
Nakamura, Yoshikazu
中科院分区：
文献类型：
--
作者：
Adachi, Hironori;Ishiguro, Akira;Nakamura, Yoshikazu
白介素17(IL-17)是一种促炎细胞因子，主要由CD4(+)细胞亚群产生，称为Th17细胞，参与宿主防御、炎症和自身免疫性疾病。IL-17A和IL-17F是结构上关系最密切的两个IL-17家族成员，它们形成同二聚体(IL-17A/A，IL-17F/F)和异二聚体(IL-17A/F)复合体。尽管IL-17A和IL-17F的生物学意义已经被用各自的抗体或基因敲除小鼠进行了研究，但由于缺乏针对IL-17A/F的特异性抑制工具，IL-17A/F异二聚体的功能研究一直受到阻碍。在本研究中，我们旨在开发一种特异性抑制IL-17A/F的RNA适配子。适体是基于与靶分子的高亲和力而在体外选择的短单链核酸序列。通过对异二聚体和同二聚体的反复选择和反选择，筛选出一种抗人IL-17A/F的适配子AptAF42。因此，AptAF42可与IL-17A/F结合，但不能与IL-17A/A或IL-17F/F结合。在体外表面等离子共振实验中，优化的衍生物AptAF42doe1可阻断IL-17A/F与IL-17受体的结合，但不能阻断IL-17A/A或IL-17F/F与IL-17受体的结合。AptAF42doe1始终阻断IL-17A/F诱导的细胞因子Gro-α的产生，但不能阻断IL-17A/A或IL-17F/F诱导的细胞因子Gro-α的产生。在IL-17A/F存在或不存在的情况下，使用核糖核酸酶针对AptAF42doe1的RNA足迹分析表明，部分预测的二级结构在替代形式之间波动，AptAF42doe1受到全局保护，不被IL-17A/F切割。这些结果表明，所选择的适体识别由IL-17A/F的异二聚体表面指定的整体构象。(C)2011 Elsevier Masson SAS。版权所有。
Interleukin-17 (IL-17) is a pro-inflammatory cytokine produced primarily by a subset of CD4(+) 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-alpha 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. (C) 2011 Elsevier Masson SAS. All rights reserved.