Detection of Single c-di-AMP by an Aerolysin Nanopore

doi:10.6023/A19060230

Acta Chimica Sinica ›› 2019, Vol. 77 ›› Issue (10): 989-992.DOI: 10.6023/A19060230 Previous Articles Next Articles

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基于Aerolysin纳米孔道对单个c-di-AMP分子的检测研究

牛红艳^a, 胡正利^b, 应佚伦^a^b^*(), 龙亿涛^a^b

^a 华东理工大学化学与分子工程学院上海 200237
^b 南京大学化学化工学院生命分析化学国家重点实验室南京 210023

投稿日期:2019-06-24 发布日期:2019-08-13
通讯作者: 应佚伦 E-mail:yilunying@nj.edu.cn
基金资助:
项目受国家自然科学基金(21922405);项目受国家自然科学基金(61871183);项目受国家自然科学基金(21834001);国家“万人计划”青年拔尖人才资助

Detection of Single c-di-AMP by an Aerolysin Nanopore

Niu, Hongyan^a, Hu, Zhengli^b, Ying, Yilun^a^b^*(), Long, Yi-Tao^a^b

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

Received:2019-06-24 Published:2019-08-13
Contact: Ying, Yilun E-mail:yilunying@nj.edu.cn
Supported by:
Project supported by the National Natural Science Foundation of China(21922405);Project supported by the National Natural Science Foundation of China(61871183);Project supported by the National Natural Science Foundation of China(21834001);the National Ten Thousand Talent Program for Young Top-notch Talent

1. Detection of Single c-di-AMP by an Aerolysin Nanopore.PDF(793KB)

Abstract

Cyclic di-AMP (c-di-AMP) is a ubiquitous second messenger in prokaryotic cells. c-di-AMP can not only effectively regulate various physiological processes such as cell growth, ion transport and cell wall metabolism balance, but also trigger type I interferon response to inspire the body's immune response. Nanopore-based single molecule detection technology is an emerging single molecule detection method which is currently applied to various fields since it has many advantages such as high speed, label-free, high sensitivity and low cost. Aerolysin is a robust biological nanopore with high temporal resolution and high current resolution, which has achieved single oligonucleotide detection, polysaccharide analysis and the studies of enzymolysis kinetics. Aerolysin nanopore is negatively-charged protein nanopore which has numerous negatively charged amino acid residues around its cis entrances. The electrostatic repulsion between the negatively charged c-di-AMP and negatively charged amino acid residues around the cis entrances prevents c-di-AMP entering the nanopore. In this study, 1.0 mol/L LiCl was used as electrolyte solution to facilitate aerolysin analysis of single c-di-AMP molecule. Each event can be characterized by two parameters, the current blockade, I/I₀, and the blockade time, τ_off. The blockades are classified into two populations as PI and PII. The PI events are assigned to c-di-AMP that bump into the pore and then diffuse away. PII events are assigned to traversing of c-di-AMP through the nanopore. Compared with potassium ions, lithium ion can be more effectively to associate with the negative charges on the aerolysin nanopore surface and reduce the electrostatic repulsion between the c-di-AMP molecule and the Aerolysin. The results showed that number of PI events in per minute was significantly increased in 1.0 mol/L LiCl. The number of PI events in per minute in LiCl is 30 times than that in KCl at 90 mV. Hence, Aerolysin nanopore can be used as an ultrasensitive single molecule sensor for cyclic dinucleotides.

Key words: c-di-AMP, cyclic dinucleotides, aerolysin, single molecule interface, biological nanopores

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Niu, Hongyan, Hu, Zhengli, Ying, Yilun, Long, Yi-Tao. Detection of Single c-di-AMP by an Aerolysin Nanopore[J]. Acta Chimica Sinica, 2019, 77(10): 989-992.

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Fig. & Tab. 3

Figure 1. (a) Single-molecule analysis of a c-di-AMP with an aerolysin nanopore. (b) The chemical structure of c-di-AMP. The current blockade traces and the typical current blocked event of c-di-AMP translocating through an aerolysin pore in 1.0 mol/L (c) LiCl and (d) KCl at 100 mV, respectively. Data were obtained with the presence of 40 mg/L c-di-AMP at the cis side of the Aerolysin nanopore

Figure 2. Two-dimensional (2D) contour plots, related I/I0 and τoff histograms for the aerolysin analysis of c-di-AMP at 100 mV in 1.0 mol/L LiCl (a) and 1.0 mol/L LiCl (b). I/I0 histograms of c-di-AMP by aerolysin were fitted to multiple Gaussian peak. τoff histograms were also fitted to Gaussian peak. The blockades of c-di-AMP are classified into two populations as PI and PII. The fitted Gaussian peaks in I/I0 histrogram is shown in Figure S1

Figure 3. (a) The current blockade traces of c-di-AMP translocating through an aerolysin pore in 1.0 mol/L LiCl at 100 mV, 120 mV and 140 mV respectively; (b) The duration of PI in 1.0 mol/L LiCl; (c) The duration of PII in 1.0 mol/L LiCl; (d) The number of PI events per minute in LiCl and KCl; (e) The number of PI/(PI+PII) per minute in LiCl and KCl

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基于Aerolysin纳米孔道对单个c-di-AMP分子的检测研究

Detection of Single c-di-AMP by an Aerolysin Nanopore

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