重组人促红细胞生成素-α DNA适配体的活性高级结构特征

doi:10.7503/cjcu20170312

高等学校化学学报 ›› 2018, Vol. 39 ›› Issue (1): 48.doi: 10.7503/cjcu20170312

重组人促红细胞生成素-α DNA适配体的活性高级结构特征

贺小琴, 何军林, 徐华, 郭磊(), 谢剑炜

抗毒药物与毒理学国家重点实验室, 军事医学科学院毒物药物研究所, 北京 100850

收稿日期:2017-05-18 出版日期:2018-01-10 发布日期:2017-11-13
作者简介:联系人简介: 郭磊, 女, 博士, 研究员, 主要从事生物分析化学和毒物药物分析研究. E-mail:guolei@bmi.ac.cn
基金资助:
国家自然科学基金(批准号: 21175152)资助

Active Conformation of DNA Aptamer Against Recombinant Human Erythropoietin-α^†

HE Xiaoqin, HE Junlin, XU Hua, GUO Lei^*(), XIE Jianwei

Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, State Key Laboratory of Toxicology and Medical Countermeasures, Beijing 100850, China

Received:2017-05-18 Online:2018-01-10 Published:2017-11-13
Contact: GUO Lei E-mail:guolei@bmi.ac.cn
Supported by:
† Supported by the National Natural Science Foundation of China(No.21175152)

摘要/Abstract

摘要：

针对重组人促红细胞生成素-α(EPO-α)的最短适配体序列In27, 使用表面等离子体共振、圆二色光谱及荧光光谱等多种技术, 研究和明确了In27的活性与构型间的关系. 结果表明, 阳离子Na⁺的存在及其浓度是影响In27产生特异性亲和识别的关键因素; 在Na⁺诱导下In27形成了平行/反平行的混合G四链体(GQ)构型; 荧光探针硫磺素T(ThT)能特异性结合该混合构型, EPO-α的加入可与ThT形成竞争, 从而便于利用ThT创建活性EPO-α的“光开关”型检测探针.

关键词: 表面等离子体共振, 圆二色光谱, 荧光探针, G-四链体, Na+

Abstract:

A series of spectroscopic techniques such as surface plasmon resonance, circular dichroism and fluorescence was employed to illustrate the relationship between the binding activity and conformation of the shortest aptamer In27 against recombinant human erythropoietin-α(EPO-α). It was found that the presence and concentration of sodium cation were the key factors that affected the specific affinity recognition of aptamer In27. Sodium cation induced aptamer In27 to form a parallel/antiparallel hybrid G-quadruplex(GQ) conformation. GQ-specific fluorescent probe thysmenonine T(ThT) specifically bound to the hybrid GQ conformation of aptamer In27. The addition of EPO-α could compete with ThT to facilitate to create a label-free fluorescent “turn-off” probe for EPO-α. This research provides an effective combination of techniques for elucidating the active conformation of novel aptamers, and provides a kind of aptameric modules with clear interaction features for analytical, sensing and diagnostic applications.

Key words: Surface plasmon resonance, Circular dichroism, Fluorescent probes, G-Quadruplex, Sodium cation

中图分类号:

O657.3

TrendMD:

贺小琴, 何军林, 徐华, 郭磊, 谢剑炜. 重组人促红细胞生成素-α DNA适配体的活性高级结构特征. 高等学校化学学报, 2018, 39(1): 48.

HE Xiaoqin, HE Junlin, XU Hua, GUO Lei, XIE Jianwei. Active Conformation of DNA Aptamer Against Recombinant Human Erythropoietin-α^†. Chem. J. Chinese Universities, 2018, 39(1): 48.

图/表 6

Fig.1 Influences of cation kind and concentration on the binding response between aptamer and EPO-α as observed in SPR a. In27; b. 39nt. Buffer: Tris-S.

Fig.2 Multiple cycle kinetics of In27 towards immobilized EPO-α in Tris-S(A) or Tris-140Na buffer(B)(A) Ka=2.85×104 L·mol-1·s-1, Kd=1.52×10-3 s-1, KD=53.3 nmol/L, Chi2=0.197, U-Value=1; (B) Ka=3.84×104 L·mol-1·s-1, Kd=2.02×10-3 s-1, KD=52.7 nmol/L, Chi2=0.779, U-Value=1.

Fig.3 CD spectra of aptamer In27 or 39nt at 5 μmol/L in Tris-S buffer(A), In27 in Tris-Na buffer(B) and In27 in Tris-K buffer(C)

Fig.4 Fluorescence spectra of ThT(5 μmol/L) with In27(4 μmol/L) in Tris-Na buffer(A) and

Fig.5 Fluorescence spectra of 5 μmol/L ThT upon titration with In27 from 0 to 10 μmol/L(A) and Job’s experiments on binding ratio of In27 and ThT(B)(A) Concentration of In27 from top to bottom/(μmol·L-1): 10, 5, 2, 1, 0.5, 0.2, 0.1, 0.05, 0; (B) the concentration of In27 or ThT was varied from 0 to 5 μmol/L to compose the molar ratio of In27 to In27+ThT between 0 to 1, both slit widths of emission and excitation were 5 nm.

Fig.6 Fluorescence spectra(A) and titration curve(B) of ThT in the presence of In27 upon addition of EPO-αBoth concentrations of In27 and ThT were 2 μmol/L. Concentration of EPO-α, from top to bottom/(μmol·L-1): 0.1, 0.25, 0.5, 2.5, 5, 10. Both slit widths of emission and excitation were 10 nm. Inset of (B) shows the linearity curve between fluorescence intensity and the concentration of EPO-α.

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重组人促红细胞生成素-α DNA适配体的活性高级结构特征

Active Conformation of DNA Aptamer Against Recombinant Human Erythropoietin-α^†

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重组人促红细胞生成素-α DNA适配体的活性高级结构特征

Active Conformation of DNA Aptamer Against Recombinant Human Erythropoietin-α†

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Active Conformation of DNA Aptamer Against Recombinant Human Erythropoietin-α^†