Aerolysin单分子界面的构建及高选择性单分子检测

doi:10.7503/cjcu20190364

高等学校化学学报 ›› 2019, Vol. 40 ›› Issue (9): 1825.doi: 10.7503/cjcu20190364

Aerolysin单分子界面的构建及高选择性单分子检测

武雪原¹,应佚伦^1,²,龙亿涛^1,^2,^*()

^1. 华东理工大学化学与分子工程学院, 上海 200237
^2. 南京大学化学化工学院, 生命分析化学重点实验室, 南京 210023

收稿日期:2019-07-01 出版日期:2019-09-10 发布日期:2019-09-09
通讯作者: 龙亿涛 E-mail:ytlong@ecust.edu.cn;yitaolong@nju.edu.cn
基金资助:
国家自然科学基金(21834001, 61871183)

Construction and High Selectivity of Aerolysin Single Molecular Interface for Single Molecule Analysis ^†

WU Xueyuan¹,YING Yilun^1,²,LONG Yitao^1,^2,^*()

^1. School of Chemistry and Molecular Engineering, East China University of Science and Technology,Shanghai 200237, China
^2. State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering,Nanjing University, Nanjing 210023, China

Received:2019-07-01 Online:2019-09-10 Published:2019-09-09
Contact: LONG Yitao E-mail:ytlong@ecust.edu.cn;yitaolong@nju.edu.cn
Supported by:
Supported by the National Natural Science Foundation of China(21834001, 61871183)

摘要/Abstract

摘要：

提出了一种基于Aerolysin膜蛋白质分子构建单分子界面的方法, 运用蛋白质工程技术对单分子界面进行定点修饰, 所建立方法灵活、可控且重复性好. 采用Poly(dA)₄为探针分子对修饰后的单分子界面进行了表征, 结果表明, 在孔口处的Arginine修饰影响了寡聚核苷酸的选择性. 为进一步理解Aerolysin单分子界面及合理设计功能性单分子界面提供了参考.

关键词: 气单胞菌溶素, 单分子界面, 纳米孔道, 单分子分析

Abstract:

As an new conceptual single molecular interface, the biomolecular nanopore can accommodate only a single molecule and contains abundant amino acid residues in the lumen, providing an appropriate confinement for single molecule detection. Herein, a generalized method is proposed for the construct of single molecular interface based on membrane protein. The method is flexible, controllable and repeatable. The characterization of D209R mutation with Poly(dA)₄ illustrate that the selectivity of aerolysin single molecular interface is related the electrostatic properties at the entrance of nanopore, which deepens our understanding of aerolysin single molecular interface, providing a paradigm for rational design of functional single molecular interface.

Key words: Aerolysin, Single molecular interface, Nanopores, Single molecule analysis

中图分类号:

O657

TrendMD:

武雪原, 应佚伦, 龙亿涛. Aerolysin单分子界面的构建及高选择性单分子检测. 高等学校化学学报, 2019, 40(9): 1825.

WU Xueyuan, YING Yilun, LONG Yitao. Construction and High Selectivity of Aerolysin Single Molecular Interface for Single Molecule Analysis ^†. Chem. J. Chinese Universities, 2019, 40(9): 1825.

图/表 4

Fig.1 Construction of prokaryotic recombinant plasmid of pET-22b-Proaerolysin

Fig.2 Construction of aerolysin-based D209R single biomolecule interface (A) Illustration of the nanopore electrochemical measurement. Poly(dA)4 are used as the probe molecule to characterize the aerolysin-based single biomolecular interface. The location of 209 sites is marked by red band; (B) the chemical structure of negative-charge aspartic acid and positive-charge arginine; (C) the characteristic blockage signals of Poly(dA)4 in WT(black) and D209R(red) aerolysin single biomolecule interface.

Fig.3 Electrochemical characterization of aerolysin-based single biomolecule interface (A) SDS-PAGE result of WT and D209R mutant proaerolysin; (B) raw current traces of Poly(dA)4 in D209R single biomolecule interface for 8 h. Poly(dA)4 were added to the cis solution at 2 h after the nanopore formation.

Fig.4 Statistical analysis of Poly(dA)4 in WT and D209R aerolysin single molecular interface (A) I-V curves of WT(black) and D209R(red) aerolysin single molecular interface. I/I0 (B), duration time(C) and frequency(D) of Poly(dA)4 vs. applied voltage ranging from 80 to 180 mV in WT(black) and D209R(red) aerolysin single molecular interface.

参考文献 36

[1]	Peng Y. Y., Qian R. C., Hafez M. E., Long Y. T ., ChemElectroChem 2017, 4( 5), 977— 985
[2]	Yang J., Li S., Wu X. Y., Long Y. T., ,Chin. J. Anal. Chem., 2017,45( 12), 1766— 1775
	(杨洁, 李爽, 武雪原, 龙亿涛.分析化学,2017,45(12), 1766— 1775)
[3]	Qiu K. P., Wu X. Y., Yang J., Ying Y. L., Long Y. T., ,. Curr. Opin. Chem. 2018, 10( 16), 46— 53
[4]	Ying Y. L., Cao C., Hu Y. X., Long Y. T., ,. Natl. Sci. Rew. 2018, 5( 4), 450— 452
[5]	Cao C., Liao D. F., Ying Y. L., Long Y. T., ,Acta Chim. Sinica, 2016,74( 9), 734— 737 doi: 10.6023/A16070352
	(曹婵, 廖冬芳, 应佚伦, 龙亿涛.化学学报,2016,74(9), 734— 737) doi: 10.6023/A16070352
[6]	Hu Z. L., Li M. Y., Liu S. C., Ying Y. L., Long Y. T., ,. Chem. Sci. 2019, 10( 2), 354— 358
[7]	Cao C., Long Y. T., ,. Acc. Chem. Res. 2018, 51( 2), 331— 341
[8]	Liao D. F., Cao C., Ying Y. L., Long Y. T ., Small 2018, 14( 18), e1704520
[9]	Li Q., Lin Y., Ying Y. L., Liu S. C., Long Y. T., ,Sci. Sin. Chim., 2017,47( 12), 1445— 1449
	(李巧, 林瑶, 应佚伦, 刘少创, 龙亿涛.中国科学: 化学,2017,47(12), 1445— 1449)
[10]	Ying Y. L., Zhang X., Liu Y., Xue M., Li H., Long Y. T., ,Acta Chim. Sinica, 2013, 71, 44— 50
	( 应佚伦, 张星, 刘钰, 薛梦竹, 李洪林, 龙亿涛 . 化学学报, 2013,71, 44— 50
[11]	Hu Z. L., Li Z. Y., Ying Y. L., Zhang J. J., Cao C., Long Y. T., ,. Anal. Chem. 2018, 90( 7), 4268— 4272
[12]	Kasianowicz J., Brandin E., Branton D., Deamer D ., Proc. Natl. Acad. Sci. USA 1996, 93( 24), 13770— 13773
[13]	Reiner J. E., Balijepalli A., Robertson J. W. F., Campbell J., Suehle J., ,. Chem. Rev. 2012, 112( 12), 6431— 6451
[14]	Oukhaled A., Bacri L., Pastoriza G. M., Betton J. M., Pelta J., ,. ACS Chem. Biol. 2012, 7( 12), 1935— 1949
[15]	Shendure J., Ji H., ,. Nat. Biotechnol. 2008, 26( 10), 1135— 1145
[16]	Asandei A., Rossini A. E., Chinappi M., Park Y., Luchian T ., Langmuir 2017, 33( 50), 14451— 14459
[17]	Manrao E. A., Derrigton I. M., Laszlo A. H., Langford K. W., Hopper M. K., Gillgren N., Pavlenok M., Niederweis M., Gundlach J. H., ,. Nat. Biotechnol. 2012, 30( 4), 349— 353
[18]	Wang Y., Tian K., Du X., Shi R. C., Gu L. Q., ,. Anal. Chem. 2017, 89( 24), 13039— 13043
[19]	Xi D. M., Li Z., Ai S. Y., Zhang S. S., ,. Anal. Chem. 2018, 90( 1), 1029— 1034
[20]	Mandal T., Kanchi S., Ayappa K. G., Maiti P. K ., Nanoscale 2016, 8( 26), 13045— 13058
[21]	Parker M. W., Buckley J. T., Postma J. P., Tucker A. D., Leonard K., Pattus F., Tsernoglou D ., Nature 1994, 367( 6460), 292— 295
[22]	Stefureac R., Long Y. T., Kraatz H. B., Howard P., Lee J. S ., Biochemistry 2006, 45( 30), 9172— 917
[23]	Wen S., Zeng T., Liu L., Zhao K., Zhao Y., Liu X., Wu H. C., ,. J. Am. Chem. Soc. 2011, 133( 45), 18312— 18317
[24]	Pastorizagallego M., Rabah L., Gibrat G., Thiebot B., Goot F. G., Auvray L., Betton J. M., Pelta J., ,. J. Am. Chem. Soc. 2011, 133( 9), 2923— 2931
[25]	Fennouri A., Daniel R., Pastoriza-Gallego M., Auvray L., Pelta J., Bacri L., ,. Anal. Chem. 2013, 85( 18), 8488— 8492
[26]	Degiacomi M. T., Iacovache I., Pernot L., Chami M., Kudryashev M., Stahlberg H., Goot F. G., Peraro M. D., ,. Nat. Chem. Biol. 2013, 9( 10), 623— 629
[27]	Ioan I., Sacha D. C., Nuria C., Matteo D. P., van der Goot F. G., Benoif Z., ,. Nat. Commun. 2016, 7, 12062
[28]	Cao C., Ying Y. L., Hu Z. L., Liao D. F., Tian H., Long Y. T., ,. Nat. Nanotechnol. 2016, 11, 713— 718
[29]	Yu J., Cao C., Long Y. T., ,. Anal. Chem. 2017, 89( 21), 11685— 11689
[30]	Wang Y. Q., Cao C., Ying Y. L., Li S., Wang M. B., Huang J., Long Y. T., ,. ACS Sens. 2018, 3( 4), 779— 783
[31]	Wang Y. Q., Li M. Y., Qiu H., Cao C., Wang M. B., Wu X. Y., Huang J., Ying Y. L., Long Y. T., ,. Anal. Chem. 2018, 90( 13), 7790— 7794
[32]	Cao C., Li M. Y., Cirauqui N., Wang Y. Q., Peraro M., Tian H., Long Y. T., ,. Nat. Commun. 2018, 9, 2823
[33]	Wu X. Y., Wang M. B., Wang Y. Q., Li M. Y., Ying Y. L., Long Y. T., ,. CCS Chem. 2019, 1( 3), 304— 312
[34]	Cao C., Liao D F., Yu J., Tian H., Long Y . T .,Nat. Protoc., 2017, 12, 1901—1911
[35]	Balijepalli A., Ettedgui J., Cornio A. T., Robertson J. W. F., Cheung K. P., Kasianowicz J. J., Vaz C., ,. ACS Nano 2014, 8, 1547— 1553
[36]	Degiacomi M. T., Iacovache I., Pernot L., Chami M., Kudryashev M., Stahlberg H., Goot F. G., Peraro M. D., ,. Nat. Chem. Biol. 2013, 9, 623— 629

Aerolysin单分子界面的构建及高选择性单分子检测

Construction and High Selectivity of Aerolysin Single Molecular Interface for Single Molecule Analysis ^†

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Aerolysin单分子界面的构建及高选择性单分子检测

Construction and High Selectivity of Aerolysin Single Molecular Interface for Single Molecule Analysis †

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Construction and High Selectivity of Aerolysin Single Molecular Interface for Single Molecule Analysis ^†