两种表征核酸适配体-靶蛋白亲和作用的毛细管电泳方法比较

doi:10.3724/SP.J.1123.2020.04001

摘要/Abstract

摘要：

适配体-靶分子间的亲和作用表征是理解和应用核酸适配体发挥特异亲和作用的基本前提，CE技术则为上述表征提供了多模式的简捷途径，但多种模式体系间的结果往往存在差异，导致CE亲和评价可靠性和进一步应用受到限制，亟须建立多CE方法测定适配体-靶分子间亲和作用的系统比较研究。该研究以凝血酶及其特异性作用于肝素结合位点的适配体29mer为模型体系，基于CE-激光诱导荧光检测，引入CE-迎头分析（FA）评价方法，并比较其与预平衡-毛细管区带电泳（PE-CZE）的异同。首先进行了CE-FA方法分离条件的优化，37 ℃、0.5 h孵育完全后进样，进样时间为30 s，在较低工作温度（15 ℃）、较短毛细管长度（30 cm）及生物相容性好的缓冲体系2×TG（Tris-甘氨酸缓冲液，pH 8.5）条件下，经15 kV分离时，得到了稳定的荧光标记29mer（F29mer）-凝血酶复合物及游离F29mer平台峰。加入1 g/L牛血清白蛋白（BSA），有效提高了CE-FA平台峰高及迁移时间的重复性。详细讨论了两种方法下6种拟合方式的结果及特点。针对CE-FA和PE-CZE法，以结合适配体/游离适配体的浓度比对游离适配体浓度非线性拟合、平台峰高变化对浓度间的非线性拟合、平衡混合物的非平衡CE（NECEEM）计算等进行拟合。结果表明，6种拟合结果中5种不存在显著性差异，得到的解离常数（K_d）值均介于24~64 nmol/L范围。CE-FA法中的3种拟合结果符合度较好，说明CE-FA法易于在非平衡的CE分离体系下保持适配体-复合物间的结合-解离平衡，所测得K_d准确度较高。PE-CZE法中，借由降低的游离F29mer峰高对凝血酶浓度间的非线性拟合所得K_d值偏差过大；选择凝血酶浓度为F29mer浓度的0.5~2倍，且观察到明显的两峰间"指数桥"为前提，可经NECEEM法进行数据计算求解得到较为准确的K_d值。CE-FA法可与PE-CZE法互为印证，提高了亲和作用评价的可靠性。首选推荐使用多浓度平台峰高变化进行非线性拟合的CE-FA法，可相对有效克服高压电场对复合物稳定性的影响，具有适用范围广、方法稳定、结果拟合简便准确等特点。

关键词: 毛细管电泳, 前沿分析, 亲和作用, 适配体, 凝血酶, 非线性拟合

Abstract:

Affinity interaction characterization is a prerequisite for understanding the specific binding of nucleic acid aptamers to their target molecules and consequently their appropriate applications. The CE technique provides a simple and multi-mode approach to such a characterization, but different results obtained from multiple modes and systems lead to limited reliability and further applications. Thus, there is an urgent need to develop systematic comparison approaches of multi-mode applications in CE, which would allow a better investigation of the affinity between aptamers and target molecules.

In this work, based on CE laser-induced fluorescence detection, we applied the CE frontal analysis (FA) approach for affinity evaluation and compared it with preequilibrium-capillary zone electrophoresis (PE-CZE) using thrombin and its 29-mer aptamer as a model system that specifically binds to the heparin binding site.

The optimization conditions of the CE-FA method included 30 s injection of a mixture incubated at 37℃ for 0.5 h followed by separation at low (15℃) working temperature using short capillary (30 cm) under 15 kV in a biocompatible buffer (2×TG, pH=8.5), which provided stable plateau peaks of complex and free fluorescent-labeled 29-mer (F29-mer) aptamers. The addition of 1 g/L bovine serum albumin (BSA) enhanced the reproducibility of the plateau peak heights and migration times during the CE-FA separation.

We then discussed in detail the results and features obtained from six fitting modes of the two methods. We applied multiple fitting modes for CE-FA and PE-CZE, including the nonlinear fitting of bound/free aptamer ratio versus free aptamer concentration, non-linear fitting of plateau peak height versus concentrations, and non-equilibrium CE of equilibrium mixtures (NECEEM) calculation. We observed that 5 of 6 fitting results showed no significant difference and all dissociation constant (K_d) values were in the range between 24 and 64 nmol/L. Three fitting modes of the CE-FA approach aligned well with each other, indicating that the association-dissociation equilibrium between the aptamer and complex could be easily maintained under the non-equilibrium CE separation conditions using the CE-FA method, and the measured K_d values were more accurate. Using the PE-CZE method, the K_d fitted from nonlinear regression through free aptamer peak height decrease versus concentration indicated a significant deviation. Moreover, we obtained accurate K_d values from the NECEEM calculation approach by choosing 0.5-2-fold thrombin against F29-mer. This approach allowed the clear observation of an exponential bridge between the two peaks in the electrophoregrams.

The CE-FA and PE-CZE methods could be used to mutually confirm each other, improving the affinity evaluation reliability. In this work, we recommend the selection of the CE-FA evaluation method as a priority, with a fitting mode through plateau peak height derived from a series of various concentrations. It could effectively address the problem of the high voltage-affected unstable complex peaks in CE. This provides such advantages that allow wide application, robust use, and feasible and accurate fitting results.

Key words: capillary electrophoresis (CE), frontal analysis (FA), affinity interaction, aptamer, thrombin, nonlinear fitting

孟庆威, 郭磊, 谢剑炜. 两种表征核酸适配体-靶蛋白亲和作用的毛细管电泳方法比较[J]. 色谱, 2020, 38(9): 1078-1084.

MENG Qingwei, GUO Lei, XIE Jianwei. Comparison of two capillary electrophoresis methods for aptamer-protein affinity characterization[J]. Chinese Journal of Chromatography, 2020, 38(9): 1078-1084.

图/表 5

图1 CE-FA分离条件的优化

Fig. 1 Optimization of capillary electrophoresis-frontal analysis (CE-FA) separation conditions a. injection time; b. separation voltage; c. added amount of bovine serum albumin (BSA).A: free F29mer; C*: complex peak; mRFU: milli-of relative fluorescence unit.

图2 F29mer-凝血酶相互作用的(a)CE-FA谱图及(b)R-Cf非线性拟合

Fig. 2 (a) CE-FA electrophoregrams of F29mer-thrombin interaction and (b) non-linear fitting by R versus Cf values Cf refers to free aptamer concentration, and R refers to ratio of Cf to the binding aptamer concentration (Cb).

图3 由(a)CE-FA法的平台峰高变化和(b)PE-CZE法谱峰峰高变化对凝血酶浓度进行非线性拟合

Fig. 3 Non-linear fitting curves of (a) plateau peak height in CE-FA and (b) peak height in preequilibrium-capillary zone electrophoresis (PE-CZE) versus concentration of thrombin A refers to the relative decrease of (plateau) peak height of free F29mer; C* refers to the relative increase of (plateau) peak height.

表1 CE-FA与PE-CZE方法下F29mer-凝血酶亲和作用参数

Table 1 Affinity parameters of F29mer-thrombin determined by CE-FA or PE-CZE methods

Method	K_d/(nmol/L)	n	r²
# Fitted via single-site binding model. /: not applicable.
CE-FA (R-C_f)	42±28	1	0.768
CE-FA (A)	24±2	1^#	0.995
CE-FA (C^*)	61±14	1^#	0.972
PE-CZE (A)	147±63	1^#	0.912
PE-CZE (C^*)	64±12	1^#	0.980
PE-CZE (NECEEM)	56±25	/	/

图4 PE-CZE模式下用于NECEEM法计算的峰-桥曲线

Fig. 4 Peak-bridge curves calculated by non-equilibrium CE of equilibrium mixtures (NECEEM) in PE-CZE mode

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