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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2019, Vol. 39 Issue (11): 96-104    DOI: 10.13523/j.cb.20191111
综述     
小分子靶标与其核酸适配体亲和力的表征方法 *
苏艺,蒋灵丽,林俊生()
华侨大学医学院 泉州 362021
Characterization of the Affinity Between Low Molecular Weight Targets and Their Aptamers
SU Yi,JIANG Ling-li,LIN Jun-sheng()
School of Medicine, HuaQiao University, Quanzhou 362021, China
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摘要：

核酸适配体是指通过SELEX筛选得到的能与靶标高特异性、高亲和力结合的单链DNA或RNA。目前国内外具有高亲和性和高特异性结合的小分子靶标的核酸适配体依然很少,究其原因,一方面是因为小分子靶标的适配体难以筛选,另一方面是小分子靶标与其候选适配体亲和力表征方法难以确定。亲和力表征是确定适配体筛选成功与否的关键步骤,就现有的小分子靶标与其相应适配体亲和力表征方法进行了总结,包括纳米金比色法、等温滴定量热法、表面等离子共振、圆二色谱法、石英晶体微天平法、微量热泳动法和SYBR Green I染料检测法等,并分析了这些方法的优缺点及改进建议,以期有助于提高适配体表征效率。
关键词： 核酸适配体;  小分子靶标;  亲和力表征    
Abstract:

Aptamers are single-stranded DNA or RNA who bind to their targets with high specificity and affinity obtained by selection from a library through SELEX. At present, there are only a few aptamers with high affinity and high specificity to low molecular weight targets all over the world. The aptamers against low molecular weight targets are difficult to be screened. Moreover, it is difficult to determine the affinity of low molecular weight targets binding with their candidate aptamers. Affinity characterization is the key step to determine whether the aptamer screening is successful or not. The existing affinity characterization methods for low molecular weight targets and their corresponding aptamers are summarized, including nanogold colorimetry, isothermal drop calorimetry, surface plasmon resonance, circular dichroism, quartz crystal microbalance, microscale thermophoresis and SYBR Green I dye detection. The advantages and disadvantages of these methods and suggestions for improvement are also discussed in order to improve the efficiency of aptamer characterization.
Key words: Aptamer    Low molecular weight target    Affinity characterization
收稿日期: 2019-04-04 出版日期: 2019-12-17
ZTFLH:  Q819  
基金资助: * 国家重点研发计划政府间国际科技创新合作重点专项(2016YFE0101700);福建省科技厅引导性项目(2016Y0063)
通讯作者: 林俊生     E-mail: junshenglin@hqu.edu.cn
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苏艺,蒋灵丽,林俊生. 小分子靶标与其核酸适配体亲和力的表征方法 *[J]. 中国生物工程杂志, 2019, 39(11): 96-104.

SU Yi,JIANG Ling-li,LIN Jun-sheng. Characterization of the Affinity Between Low Molecular Weight Targets and Their Aptamers. China Biotechnology, 2019, 39(11): 96-104.

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https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20191111    或    https://manu60.magtech.com.cn/biotech/CN/Y2019/V39/I11/96

方法 基本原理 靶标案例 Kd值 适用范围 优势 不足 参考文献
纳米
金法		 普通比色法:纳米金聚集与分散状态颜色变化 啶虫脒 5nmol/L 靶标分子本身为无色 现象明显易制备,操作简单,耗时短 普适性不强,部分小分子靶标甚至会促进适配体更加稳定地吸附在纳米金上而出现相反的检测结果 [4]
妥布霉素 23.3nmol/L [5]
钾离子 0.42nmol/L [6]
三聚氰胺 14.9nmol/L [7]
荧光猝灭活性 赭曲霉毒素A 5nmol/L [8]
雌二醇 0.48nmol/L [9]
过氧化氢酶样活性 玉米烯酮 10ng/ml [10]
磺胺二甲氧嘧啶 10ng/ml [11]
ITC 检测靶标与适配体结合时热量变化 L-酪氨酰胺 16.8μmol/L 范围较广 精确度高,实时连续监测,可直接测得Kd值 用样量较大,低通量,需专人操作 [12]
替硝唑 1nmol/L [13]
孔雀石绿 (50±20)nmol/L [14]
SPR 检测适配体与靶标结合前后光折射率变化 妥布霉素 (1.24±0.03)
nmol/L		 需固定适配体或者靶标 灵敏度高,实时性号,芯片可再生 小分子检测信号低,小分子难固定、低通量 [15]
黄曲霉毒素1 0.94ng/ml [16]
氟乙酰胺 1μmol/L [17]
CD 检测适配体与靶标结合前后左旋及右旋光变化 25-羟基
维生素D3		 11nmol/L 适配体与靶标结合前后结构变化明显 快速、高效反映适配体结构变化 样品制备时纯度等要求高、前处理复杂 [18]
QCM 适配体与靶标结合前后重量变化,转化为频率变化检测 茶碱 0.526μmol/L 需固定适配体或者靶标 灵敏度高、实时性强 检测易受温度、湿度和震动等环境因素影响 [19]
L -酪氨酰胺 (181±27)
μmol/L		 [20]
MST 靶标与适配体在毛细管中通过温度梯度热运动,检测反应体系中荧光分布的变化 17β-雌二醇 98nmol/L 待测物分子大小不限 样品量较少、可测量天然状态下适配体与靶标之间的结合 适配体需要修饰荧光,荧光寿命短 [21]
ATP (31±3)
μmol/L		 [22]
SGI SYBR Green I绿色荧光染料,检测结合前后荧光变化 ATP 23.4nmol/L 靶标无荧光 简单、易操作 检测范围小、低通量 [23]
赭曲霉毒素A (370±250)
nmol/L		 [24]
四环素 0.10μg/ml [25]
呋喃酮 (1.1±0.4)
μmol/L		 [26]
表1  小分子靶标与其适配体亲和力检测方法总结
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