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Selective and Sensitive Detection of Methylcytosine by Aerolysin Nanopore under Serum Condition Jie Yu, Chan Cao, and Yi-Tao Long Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03133 • Publication Date (Web): 09 Oct 2017 Downloaded from http://pubs.acs.org on October 10, 2017
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Analytical Chemistry
Selective and Sensitive Detection of Methylcytosine by Aerolysin Nanopore under Serum Condition Jie Yu, Chan Cao and Yi-Tao Long* *E-mail:
[email protected]. Phone: +86-021-6425-2339 Key Laboratory for Advanced Materials & School of Chemistry and Molecular Engineering, East China University of Science and Technology, Shanghai, 200237, P. R. China. Methylated DNA, Quantification, Serum, Aerolysin, Nanopore ABSTRACT: Detection of DNA methylation in real human serum is of great importance to push the development of clinical research and early diagnosis of human diseases. Herein, taking advantage of stable pore structure of aerolysin in harsh environment, we distinguish methylated cytosine from cytosine using aerolysin nanopore in human serum. Since wild-type (WT) aerolysin enables high sensitivity detection of DNA, the subtle difference between methylated cytosine and cytosine could be measured directly without any specific designs. Methylated cytosine induced a population of I/I0 = 0.53 while cytosine was focused on I/I0 = 0.56. The dwell time of methylated cytosine (5.3 ± 0.1 ms) was much longer than that of cytosine (3.9 ± 0.1 ms), which improves the accuracy for the discrimination of the two oligomers. Moreover, the pore-membrane system could remain stable for more than 2 hours and achieve the detection of methylated cytosine with zero-background signal in the presence of serum. Additionally, event frequency of methylated cytosine is in correspondence with the relative concentration and facilitate the quantification of methylation.
DNA methylation is a crucial epigenetic modification in mammalian genomes which mainly occurs at the fifth position of cytosine.1 In mammals, DNA methylation is fundamental for gene expression2,3 and is associated with human development4 and diseases such as cancer.5,6 The detection of methylation is therefore significant to understand the influence of epigenetics and to provide new insights into clinical diagnosis. Nanopore technique is a research tool for single-molecule detection on DNA sequencing7,8 and medical diagnosis.9,10 Biological nanopores such as α-hemolysin11,12 and MspA13,14 have been employed for the discrimination and quantification of methylated cytosine without label and amplification. A more recent work provided a direct method to discriminate modified DNA by monitoring the stability of metal ion-DNA interactions.15 However, the detection of methylated DNA in serum was rarely discussed using nanopore. As commonly used in diagnostic trials, serum is qualified to play the role of background in detection. Due to the instability of lipid bilayer caused by the addition of serum, researchers tend to choose ideal experimental conditions which only contain pure analytes and buffer solutions.16 This will retard the development of nanopore sensor in clinical applications. Herein, we detect two DNA oligomers with only single methylcytosine difference using aerolysin nanopore in normal human serum. Aerolysin is a heptameric protein pore with a narrower constriction (~1.0 nm)17 compared to α-hemolysin (~1.4 nm)18 and MspA (~1.2 nm).19 Not only does smaller pore diameter guarantee high spatial resolution for aerolysin in the detection of DNA molecules, but positively charged amino acids in its lumen are also natural “brakes” that slow down the DNA translocation. We have already found that aerolysin is able to reduce the translocation speed of ssDNA by three or-
ders of magnitude (2.0 ms/nt).20-21 Thus, aerolysin is sensitive enough to detect methylated DNA without any additional methods. By monitoring and analyzing ion current, methylated cytosine was able to be distinguished from unmethylated cytosine directly. Not only the current blockade but also the dwell time was definitely well distinguished. In terms of pore structure, it is quite easy for monomeric aerolysin to form a stable pore rapidly. Moreover, aerolysin is stable under harsh conditions. For instance, aerolysin can endure electrolyte solutions over a broad pH range from 4.0 to 10.020 as well as the presence of denaturing agent.22 To further test the methylated cytosine in complex samples and to explore the possibility of aerolysin nanopore for clinical diagnosis, we detected the methylated cytosine in the presence of human serum. We showed that methylated cytosine was able to be distinguished from unmethylated cytosine in serum samples. EXPERIMENTAL SECTION: Chemicals and Reagents. Reagents were purchased from the following sources: potassium chloride (≥99%), decane (anhydrous, ≥99%) and trypsin-EDTA (Sigma-Aldrich); 1,2diphytanoyl-sn-glycero-3-phosphocholine (Avanti Polar Lipids); proaerolysin (Aerohead Scientific); ethylenediaminetetraacetic acid (EDTA, ≥99%, Aladdin Chemistry); Tris (hydroxymethyl) aminomethane (≥99%, Aladdin Chemistry). Synthetic oligonucleotides including 5’-CAAA-3’ and 5’mCAAA-3’ (Sangon Biotech). Human serum samples were obtained from The Second Military Medical University and stored at -20 °C. Single Channel Recording. The approach of inserting a single heptameric nanopore into a lipid bilayer was described
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Analytical Chemistry
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previously.23 In short, a phospholipid bilayer was formed on a 50 µm diameter aperture in a delrin cup (Warner Instruments) which separates the chamber into two compartments (cis and trans). Both the two compartments contained electrolyte solution (1 mL: 1 M KCl, 10 mM Tris, 1.0 mM EDTA, pH 8.0) while aerolysin monomers and DNA oligomers were added into cis side. Two matched Ag/AgCl electrodes, with the cis electrode connected to ground, were immersed in each chamber. For a rapid collection of high quantity data, the final concentration of each DNA oligomers was 5 µM. Target enrichment or smaller volume chamber could meet the demand of detection for a lower quantity of sample in clinical application. Effective method using polycationic probe could enrich target DNA around the nanopore and simultaneously detect DNA.24 In different concentration ratio experiments, DNA were premixed at relative molar concentrations of mC-DNA: C-DNA = 0:1, 1:0, 1:1, 2:1, 3:1 and 3:2. The final concentration of each ratio was 5 µM. In the experiments with serum samples, real human serum was added into both compartments and the final concentration was 1% (v/v). In all reported experiments, the room temperature was 21 ± 2 °C unless stated otherwise. Data Analysis. Current traces were recorded on a patch clamp amplifier (Axopatch 200B, Molecular Devices) coupled with a Digidata 1440A analog-digital converter (Molecular Devices). The events were filtered at 5 kHz and sampled at 100 kHz by a computer equipped with Clampex 10.4 software (Molecular Devices). An ultra-low noise instrument system for the highly sensitive nanopore detection has been developed recently.25 Data were processed by Clampfit 10.4 (Molecular Devices), MOSAIC26,27 and Origin software (OriginLab Corporation). The minimal requirement for data analysis was an exclusion of events that dwell times
