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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  2022, Vol. 42 Issue (8): 1-12    DOI: 10.13523/j.cb.2205010
研究报告     
基于分子模拟的河鲀毒素核酸适配体的连续优化*
颜志超,宋梦华,刘建平,黄强**()
复旦大学生命科学学院 上海 200438
Molecular Simulation-based Continuous Optimization of Nucleic-acid Aptamers Against Tetrodotoxin
YAN Zhi-chao,SONG Meng-hua,LIU Jian-ping,HUANG Qiang**()
School of Life Sciences, Fudan University, Shanghai 200438, China
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摘要：

河鲀毒素(tetrodotoxin, TTX)是一种生物碱类神经毒素,其中毒事件在世界范围内广泛发生,严重危害到人类健康,但尚无特效解毒剂,因此TTX的检测对食品安全领域有重大意义。为了得到更高效的TTX识别元件,在分子模拟指导下对SELEX筛选所得的核酸适配体TTX-27进行了连续优化。首先,使用Mini-hairpin结构替换阻碍TTX结合的茎环结构,使TTX更易与截短型适配体结合,然后将T39、C40碱基突变为C39、T40碱基,并对C39进行了2'-OH修饰,以增强结合区域碱基与TTX的氢键作用和范德瓦耳斯相互作用。微量热泳动(microscale thermophoresis, MST)实验证实,经截短、碱基突变和化学修饰的各适配体变体的亲和力均有提高,其中化学修饰变体TTX-D2-X-R结合TTX的解离平衡常数Kd为1.08 nmol/L,相较于TTX-27的亲和力提高了75.5倍。表明基于分子模拟的截短-突变-化学修饰是核酸适配体post-SELEX优化的有效途径,所得的适配体变体TTX-D2-X-R在TTX检测领域有着潜在的应用价值。
关键词： 河鲀毒素;  核酸适配体;  分子模拟;  碱基截短;  碱基突变;  化学修饰    
Abstract:

Tetrodotoxin (TTX) is an alkaloid neurotoxin, and its poisoning cases occurr worldwide and therefore seriously threat human health. However, there is no specific antidote yet for TTX, so the detection of TTX is of great importance in the field of food safety. To obtain a more efficient TTX recognition element, guided by molecular simulations, a DNA aptamer (TTX-27) previously discovered by SELEX screening was continuously optimized. First, the stem-loop structure, which hinders the TTX binding, was replaced with a mini-hairpin structure to make the TTX bind more easily to the truncated aptamer; next, T39 and C40 bases were mutated to C and T bases, respectively, and C39 was also modified with 2'-OH to enhance the hydrogen bonding and van der Waals interactions of the bases with TTX. Microscale thermophoresis (MST) experiments confirmed that the affinities of the aptamer variants were increased by the truncation, base mutation and chemical modification. The dissociation equilibrium constant Kd of the binding of the chemically modified variant TTX-D2-X-R to TTX was 1.08 nmol/L, which increased 75.5 times compared to that of TTX-27. Thus, this study demonstrates that the molecular simulation-based truncation-mutation-chemical modification is an effective approach to the post-SELEX optimization of nucleic-acid aptamers, and the resulting aptamer variant TTX-D2-X-R has potential applications in the field of TTX detection.
Key words: Tetrodotoxin    Aptamer    Molecular simulation    Base truncation    Base mutation    Chemical modification
收稿日期: 2022-05-05 出版日期: 2022-09-07
ZTFLH:  Q81  
基金资助: * 国家科技重大专项“重大新药开发”课题(2018ZX09J18112);国家自然科学基金(31971377);国家自然科学基金(31671386)
通讯作者: 黄强     E-mail: huangqiang@fudan.edu.cn
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引用本文:

颜志超,宋梦华,刘建平,黄强. 基于分子模拟的河鲀毒素核酸适配体的连续优化*[J]. 中国生物工程杂志, 2022, 42(8): 1-12.

YAN Zhi-chao,SONG Meng-hua,LIU Jian-ping,HUANG Qiang. Molecular Simulation-based Continuous Optimization of Nucleic-acid Aptamers Against Tetrodotoxin. China Biotechnology, 2022, 42(8): 1-12.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2205010    或    https://manu60.magtech.com.cn/biotech/CN/Y2022/V42/I8/1

图1  TTX-27 的二级(a)和三级(b)结构
名称 碱基序列(5'-3') 碱基数
TTX-27 ATAGGAGTCACGACGACCAGCTCCATTTATTCTAAATTTCATAGACTAGTTAATAATAACTATGTGCGTCTACCTCTTGA 80
TTX-D2 ATAGGAGTCACGACGACCAGCTCCATTTATTCTAAATTTCATAGACCGAAAGCGGTCTACCTCTTGA 67
TTX-D2-X ATAGGAGTCACGACGACCAGCTCCATTTATTCTAAATTCTATAGACCGAAAGCGGTCTACCTCTTGA 67
TTX-D2-X-R ATAGGAGTCACGACGACCAGCTCCATTTATTCTAAATTr(C)TATAGACCGAAAGCGGTCTACCTCTTGA 67
表1  核酸适配体序列
图2  TTX-27∶TTX复合物的RMSD值(a) 及TTX-27和TTX自发结合模拟图(b)
图3  基于残基的TTX-27截短优化前后相互作用分析图
名称 碱基序列(5'-3') 结合自由能
ΔG/(kcal/mol)
TTX-27 ATAGGAGTCACGACGACCAGCTCCATTTATTCTAAATTTCATAGACTAGTTAATAATAACTATGTGCGTCTACCTCTTGA -14.94
TTX-5S ATAGGAGTGCTCCATTTATTCTAAATTTCATAGACTAGTTAATAATAACTATGTGCGTCTACCTCTTGA -16.12
TTX-3S ATAGGAGTCACGACGACCAGCTCCATTTATTCTAAATTTCATAGACTGTGCGTCTACCTCTTGA -15.21
TTX-D1 ATAGCGAAAGCGCATTTATTCTAAATTTCATAGACTAGTTAATAATAACTATGTGCGTCTACCTCTTGA -15.89
TTX-D2 ATAGGAGTCACGACGACCAGCTCCATTTATTCTAAATTCTATAGACCGAAAGCGGTCTACCTCTTGA -17.72
表2  TTX-27变体对接能量预测值
图4  TTX-27及变体的分子模拟结果分析图和微量热泳动实验结果图
原有
碱基		 突变
碱基		 一轮突变
ΔG /(kcal/mol)		 二轮突变
ΔG/(kcal/mol)		 三轮突变
ΔG/(kcal/mol)
T38 A -6.36 -8.07 -7.25
G -5.11 * *
C * * *
T39 A -6.69 -7.78 C
G -5.11 -7.37
C -7.77 -8.75
C40 A -6.90 T T
G -8.05
T -8.20
C61 A * * *
G -6.49 -6.97 *
T * * *
T62 A -7.62 -7.65 -7.14
G -7.05 -7.52 -7.35
C -7.34 -7.16 -6.74
C63 A -5.94 -7.83 -8.69
G -7.13 -7.94 -6.42
T * * -6.95
表3  多轮突变结合自由能值
图5  基于残基的TTX-D2-X化学修饰前后相互作用分析图
碱基名称 距离/Å
C39 3.4
A41 7.1
C60 7.2
C61 6.3
C63 3.7
表4  各结合碱基脱氧核糖C-2与TTX距离值
图6  TTX-27连续优化前后与TTX的分子间相互作用
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