Subscriber access provided by Kaohsiung Medical University
Article
Simultaneous Discrimination of Single-Base Mismatch and Full Match Using a Label-Free Single-Molecule Strategy Qiufang Yang, Tingting Ai, You Lv, Yuqin Huang, Jia Geng, Dan Xiao, and Cuisong Zhou Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01285 • Publication Date (Web): 06 Jun 2018 Downloaded from http://pubs.acs.org on June 6, 2018
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 6 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Analytical Chemistry
Simultaneous Discrimination of Single-Base Mismatch and Full Match Using a Label-Free Single-Molecule Strategy Qiufang Yang,† Tingting Ai,† You Lv,† Yuqin Huang,† Jia Geng, ‡ Dan Xiao,† and Cuisong Zhou*,† †
College of Chemistry, Sichuan University, Chengdu 610064, P. R. China Department of Laboratory Medicine, West China Hospital, Sichuan University and Collaborative Innovation Center, Chengdu 610041, P. R. China
‡
KEYWORDS: simultaneous discrimination, single-base mismatch, nanopore, molecular beacon, signal-to-background ratio. ABSTRACT: Identification of single-base mismatches has found wide applications in disease diagnosis, pharmacogenetics and population genetics. However, there is still a great challenge in the simultaneous discrimination of single-base mismatch and full match. Combined with a nanopore electrochemical sensor, a shared-stem structure of molecular beacon (MB) was designed that did not need the labeling, but achieved an enhanced signal-to-background ratio (SBR) of ~104, high thermodynamic stability to bind with target sequences and a fast hybridization rate. Fully matched and single-base mismatched sequences were simultaneously discriminated at the single-molecule level, which is expected to have potential applications ranging from the quick detection, early clinical diagnostics to point-of-care research.
INTRODUCTION Because single nucleotide mutations are closely related to diseases, identification of single-base mismatches has great significance for early disease diagnosis, pharmacogenetics and population genetics.1,2 Full matched target and single-base mismatch often simultaneously coexist in cancer cells. They have high similarity in sequence but great difference in biological functions, which is a great challenge for their simultaneous identification.3,4 Although sequencing is adequate for identification of single-base mismatches, the time-costing, sophisticated instruments and rigorous experimental skills are necessary.5 Recently, polymerase chain reaction (PCR)6 and hybridization-based methods based on colorimetric,7,8 fluorescent9,10 or electrochemical11,12 signal have been proposed. These single-signal response strategies are very sensitive, but have the drawbacks of relative low specificity, false positive and tedious labeling procedures. Especially, it is difficult for the single-signal to achieve simultaneous discrimination of single-base mismatch and full match. Recent years have seen rising interest in the development of nanopore sensors, a new class of single-molecule analytical technology because of its high temporal-spatial resolution.13-15 The bacterial protein α-hemolysin (α-HL) can self-assemble across a planar lipid bilayer and form a transmembrane nanopore with the diameter of ~1.4 nm.16 Owing to its size-selective properties, α-HL nanopore allows the translocation of single nucleic acid strand but not duplex. Under an applied potential, characteristic electric signals are recorded such as the magnitude of the ionic current and the duration time, which can reveal dynamic translocation process of a single strand within a nanopore. Some nanopore sensors with a nucleic acid probe have been reported to specifically detect disease-associated miRNA17-18 and pathogenic DNA biomarker.19,20 Other nanopore sensors have been explored to
examine the unzipping kinetics of duplex DNA21 including oxidized lesions,22 DNA abasic sites23 and the unfold of G-quadruplexes hybrid.24 Recently, more and more nanopore sensors have been explored to detect single-base mismatch. For instance, Howorka et al. reported a nanopore-based sensor for the single-base mismatch, which performed 2 orders of magnitude duration difference from full-matched DNA strand.25 Xi et al. reported the detection of single-base mismatch with a 10 folds duration difference for full-matched microRNAs.26 However, there is no report to simultaneously discriminate similar sequences with single-base mismatch. The main challenge is how to enhance the specificity for sensing target sequence. To address this issue, with the combination of thermodynamic and kinetic theory studies, a shared-stem structure molecular beacon (MB) was designed that performed a high signal-to-background ratio (SBR) of ~104 for sensing its fully matched target sequence. Mixture of single-base mismatched and fully matched target were simultaneously discriminated at the single-molecule level. The label-free single-molecule strategy has great potential in early disease diagnosis, pharmacogenetics and population genetics.
EXPERIMENTAL SECTION Materials and Reagents The α-HL was purchased from Sigma-Aldrich (St. Louis, MO, USA) and used without further purification. Lipid 1, 2 - diphytanoyl - sn - glycero - 3 phosphocholine (DPhPC) was purchased from Avanti Polar Lipids Inc. (Alabaster, AL, USA). Decane was purchased from Aladdin (Shanghai, China). Teflon chamber with a 50-µm orifice was obtained from Warner Instruments (Hamden, CT, USA). All DNA oligonucleotides were synthesized by Shanghai Sangon Biotechnology Co. Ltd. (Shanghai, China).
1 ACS Paragon Plus Environment
Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Each MB was heated at 95 °C for 5 min and gradually cooled to room temperature for an hour before nanopore testing. The electrolyte solution used in this study was prepared in ultrapure water (18.2 MΩ), which contained 1.0 M KCl, 10 mM Tris-HCl and 1.0 mM EDTA (pH 8.0). Single-Channel Recording Planar lipid bilayer membranes by DPhPC were formed by applying diphytanoyl phosphatidylcholine (30 mg/mL) in decane to a 50-µm orifice in a 1 mL bilayer chamber filled with electrolyte solution. The α-HL protein (5µg/mL) was added into the cis chamber, which was connected to “ground”. The potential was applied at +120 mV. Once a single α-HL nanopore was inserted in the lipid bilayer, the mixture of MBs (final concentration of 200 nM) and target DNA (final concentration of 200 nM) were added to the cis chamber. Currents were recorded with a patch clamp amplifier (Axopatch 200B, Axon Instruments, Foster city, CA, USA). The signal was low-pass filtered at 5 kHz and sampled at a frequency of 100 kHz with a Dig data 1550A A/D converter (Axon Instruments, Foster City, CA, USA). All the recordings were performed at 22 ± 1°C. Data Analysis Data were analyzed with NANOPORE ANALYSIS which is designed by MATLAB (R2011b, MathWorks) software and Origin Lab 9.0 (OriginLab Corporation, North-ampton, MA, USA). We describe the normalized current blockages as I/I0, where I0 is the open pore current and I is the current blockages generated by the translocation of the analytes. Events with I/I0 larger than 70% were counted, which were attributed to the polymer translocation through the nanopore. Events with duration time
Page 2 of 6
longer than 1 ms were analyzed as hairpin unzipping process, which were well separated from translocation events of single strands DNA (
