Directing a rational design of aptamer-based fluorescence anisotropy assay for sensitive detection of immunoglobulin E by site-specific binding study

CORC > 生态环境研究中心 > 中国科学院生态环境研究中心 > 环境化学与生态毒理学国家重点实验室

	Directing a rational design of aptamer-based fluorescence anisotropy assay for sensitive detection of immunoglobulin E by site-specific binding study
	Zhao, Qiang; Bai, Yunlong; Wang, Hailin
刊名	TALANTA
	2020-09
卷号	217 页码:1-8
关键词	Affinity binding Binding sites Fluorescence anisotropy Fluorescence polarization Molecular interactions Sensors
ISSN号	0039-9140
英文摘要	Mapping aptamer-protein interactions is important for characterization and applications of aptamers against proteins. We describe here probing affinity interactions between aptamer and immunoglobulin E (IgE) with a fluorescence anisotropy (FA) approach using a series of aptamer probes having single fluorescein (FAM) label at individual nucleotide (A, C, T). Studies of binding between IgE and aptamer probes revealed several possible close-contact sites, e.g., T9, T10, T11, T13, C15, and T17 of a 37-nt aptamer with a stem-loop secondary structure. FAM labeling on these sites resulted in much higher FA values (higher than 0.230 for T10, T11, T13 and C15) of aptamer-IgE complexes than the distant sites (e.g., terminals) of aptamer probably because the bound IgE close to these sites significantly restricted local rotation of FAM. Close-contact site labeled aptamer probes with high affinity allowed to develop a more sensitive FA assay for IgE than distant site labeled aptamers. The FA assay using T10-labeled aptamer with a dissociation constant (K-d) about 0.8 nM enabled selective detection of IgE at 20 pM and large FA increase upon IgE addition. We also found A12, C14, A25, and T27 were important for IgE-aptamer binding as FAM labeling at these sites significantly reduced aptamer affinity. FA study showed the loop region of this stem-loop aptamer was crucial for affinity binding, and IgE bound to the loop. This FA method will be helpful for understanding aptamer-protein binding and making a rational design of aptamer affinity assays for proteins.
内容类型	期刊论文
源URL	[http://ir.rcees.ac.cn/handle/311016/44657]
专题	生态环境研究中心_环境化学与生态毒理学国家重点实验室
推荐引用方式 GB/T 7714	Zhao, Qiang,Bai, Yunlong,Wang, Hailin. Directing a rational design of aptamer-based fluorescence anisotropy assay for sensitive detection of immunoglobulin E by site-specific binding study[J]. TALANTA,2020,217:1-8.
APA	Zhao, Qiang,Bai, Yunlong,&Wang, Hailin.(2020).Directing a rational design of aptamer-based fluorescence anisotropy assay for sensitive detection of immunoglobulin E by site-specific binding study.TALANTA,217,1-8.
MLA	Zhao, Qiang,et al."Directing a rational design of aptamer-based fluorescence anisotropy assay for sensitive detection of immunoglobulin E by site-specific binding study".TALANTA 217(2020):1-8.