毛细管电泳法研究核酸库容量对蛋白质适配体筛选的影响

引用本文: 李林森, 朱超, 赵毅, 杨歌, 屈锋. 毛细管电泳法研究核酸库容量对蛋白质适配体筛选的影响[J]. 分析化学, 2020, 48(5): 615-622. doi: 10.19756/j.issn.0253-3820.191693 shu

Citation: LI Lin-Sen, ZHU Chao, ZHAO Yi, YANG Ge, QU Feng. Investigation of Library Input on Aptamers Selection Efficiency Using Capillary Electrophoresis[J]. Chinese Journal of Analytical Chemistry, 2020, 48(5): 615-622. doi: 10.19756/j.issn.0253-3820.191693 shu

毛细管电泳法研究核酸库容量对蛋白质适配体筛选的影响

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
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: 毛细管电泳（CE）具有高分辨、快速分离、样品用量小和无需固相介质等优点，是高效筛选核酸适配体的方法之一。然而，由于CE的进样量少，有限的核酸库容量是否影响毛细管电泳-指数富集配体系统进化（CE-SELEX）适配体的筛选效率，尚不明确。本研究以神经元特异性烯醇化酶（NSE）为模式蛋白，考察了CE筛选中核酸库容量对筛选效率的影响，以明确CE-SELEX方法的有效性。分别使用4个浓度（0.1、1、10和100 μmol/L）的初始核酸库进行适配体筛选，比较了初始库与经3轮筛选后的次级核酸库的亲和力。结果表明，4个核酸库筛选轮次间库亲和力相近，且经2轮筛选后均得到了微摩尔级别的核酸适配体。此外，用高浓度核酸库筛选获得的序列更具多样性，但未能提升候选序列亲和力。经2轮筛选得到NSE的核酸适配体Seq qN-01，其与NSE复合物的解离常数（K_D）为（4.72±0.15） μmol/L，并表现出很好的特异性。综上，核酸库容量会提高后续富集库的序列多样性，但对适配体筛选效率没有显著影响。

关键词:

English

Investigation of Library Input on Aptamers Selection Efficiency Using Capillary Electrophoresis

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
Received Date: 25 November 2019
Revised Date: 20 January 2020
Fund Project:
This work was supported by the National Natural Science Foundation of China (Nos. 21675012, 21874010, 21827810).

Abstract: Capillary electrophoresis (CE) is a high-efficientcy method for aptamer selection, which exhibits many merits of high resolution, rapid separation, little sample consumption, and no immobilization. However, the influence of CE injection input on aptamer systematic evolution of ligands by exponential enrichment (SELEX) is controversial because the low injection volume may result in an insufficient capacity of the oligonucleotides library. In this research, Neuron-specific enolase (NSE) was used as a model protein to investigate the effects of library input capacity on aptamer selection for clarifying the validity of CE-SELEX. Four different concentrations of initial libraries (0.1, 1, 10 and 100 μmol/L) were utilized into aptamer selection. The affinity of the initial library and sub-libraries after three rounds of selection were evaluated. The results showed that there was no significant difference in the affinity of four different sub-libraries, and the aptamers with micromolar K_D were all obtained after two rounds of selection. In addition, the sequences obtained via high concentration ssDNA library screening were more diverse, but the affinity of them was not enriched. The NSE aptamer Seq qN-01 obtained by two rounds of selection had a dissociation constant (K_D) of (4.72±0.15) μmol/L, and also presented a good specificity. In conclusion, library input capacity could increase the sequence diversity but had no considerable effects on the efficiency of aptamer selection.

Key words:

Neuron-specific enolase
/ Library input
/ Aptamer selection
/ Capillary electrophoresis-systematic evolution of ligands by exponential enrichment

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沈阳化工大学材料科学与工程学院沈阳 110142

毛细管电泳法研究核酸库容量对蛋白质适配体筛选的影响

English

Investigation of Library Input on Aptamers Selection Efficiency Using Capillary Electrophoresis

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