毛细管电泳法筛选脱铁转铁蛋白的核酸适配体及筛选影响因素分析

引用本文: 杨歌, 赵毅, 韩诗邈, 朱超, 黄渊余, 屈锋. 毛细管电泳法筛选脱铁转铁蛋白的核酸适配体及筛选影响因素分析[J]. 分析化学, 2020, 48(5): 632-641. doi: 10.19756/j.issn.0253-3820.191694 shu

Citation: YANG Ge, ZHAO Yi, HAN Shi-Miao, ZHU Chao, HUANG Yuan-Yu, QU Feng. Screening Aptamer of Apo-transferrin via Capillary Electrophoresis-Systematic Evolution of Ligands by Exponential Enrichment and Environmental Factors Analysis[J]. Chinese Journal of Analytical Chemistry, 2020, 48(5): 632-641. doi: 10.19756/j.issn.0253-3820.191694 shu

毛细管电泳法筛选脱铁转铁蛋白的核酸适配体及筛选影响因素分析

杨歌 ^1,2, ,
赵毅 ^1, ,
韩诗邈 ^1, ,
朱超 ^1, ,
黄渊余 ^1,2, ,
屈锋 ^1,

1.
北京理工大学生命学院, "分子医学与生物诊疗"工业和信息化部重点实验室, 北京 100081;
2.
北京理工大学前沿交叉科学研究院, 北京 100081
基金项目:
本文系国家自然科学基金项目（Nos.21675012，21874010，21827810）资助

摘要: 核酸适配体是通过指数富集配体系统进化（SELEX）技术，在体外筛选获得的与目标分子有高亲和力与特异性的寡核苷酸序列。由于筛选过程周期长且影响因素复杂，使用常规筛选方法进行多因素影响的条件优化的工作量大，样品消耗多，目前还少有系统的研究报道。毛细管电泳（CE）具有高分辨率、快速分离、样品用量小、筛选成本低的优势，是核酸适配体筛选的有效方法。本研究以人血清中脱铁转铁蛋白（A-TF）为模式蛋白，利用CE方法研究核酸库长度、孵育温度、缓冲溶液的种类与pH值、金属离子等因素对靶蛋白与核酸库相互作用的影响。结果表明，较短序列的核酸库与靶蛋白的亲和力更高；孵育温度、缓冲液种类与pH值均影响靶蛋白与核酸复合物的形成；低浓度的K^＋、Ca²⁺与Mg²⁺可促进复合物形成。基于优化的筛选条件，经过3轮毛细管电泳法筛选，获得了A-TF的适配体Seq A3，采用CE-LIF测得复合物的亲和常数K_D=0.476 μmol/L。此适配体可用于人血清基质中A-TF的识别。

关键词:

English

Screening Aptamer of Apo-transferrin via Capillary Electrophoresis-Systematic Evolution of Ligands by Exponential Enrichment and Environmental Factors Analysis

1.
School of Life Science, Beijing Institute of Technology, Key Laboratory of Molecular Medicine and Biological Diagnosis, Ministry of Industry and Information Technology, Beijing 100081, China;
2.
Advanced Research Institute of Multidisciplinary Science, Beijing Institute of Technology, Beijing 100081, China
Received Date: 25 November 2019
Revised Date: 12 December 2019
Fund Project:
This work was supported by the National Natural Science Foundation of China (Nos. 21675012, 21874010, 21827810).

Abstract: Aptamer is oligonucleotide with high affinity and specificity toward the target obtained via systematic evolution of ligands by exponential enrichment (SELEX). However, due to the complexity of environmental factors and the unknown influence on the selection and application of aptamers, the optimization of multiple environmental factors through conventional experiments requires a huge amount of work and samples, so there are few systematic studies. Capillary electrophoresis (CE)-SELEX has become a highly efficient technology for aptamers screening because of the advantages of CE in microscale, high efficiency and low cost. In this study, Human apo-transferrin (A-TF) was used as a model to study the effects of factors based on CE, including length of ssDNA library, incubation temperature, type and pH of incubation buffer, metal ions etc. The results showed that the shorter ssDNA library had higher affinity with the target protein, the incubation temperature, type and pH of incubation buffer affected the formation of complex, and the low concentrations of K^＋, Ca²⁺ and Mg²⁺ metal ions promoted the formation of complexes. The A-TF aptamer Seq A3 obtained by three rounds of selection had a dissociation constant (K_D) of 0.476 μmol/L, and its affinity and specificity were verified by CE-laser induced fluorescence in human serum environment. The results indicated that Seq A3 could specifically recognize A-TF in serum.

Key words:

Apo-transferrin
/ Aptamer screening
/ Influencing factors
/ Capillary electrophoresis
/ Systematic evolution of ligands by exponential enrichment

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毛细管电泳法筛选脱铁转铁蛋白的核酸适配体及筛选影响因素分析

English

Screening Aptamer of Apo-transferrin via Capillary Electrophoresis-Systematic Evolution of Ligands by Exponential Enrichment and Environmental Factors Analysis

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