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Discrimination Cascade Enabled Selective Detection of Single-Nucleotide Mutation Lidan Li, Xianjin Xiao, Jingyang Ge, Manli Han, Xu Zhou, Lei Wang, Xin Su, and Changyuan Yu ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.7b00005 • Publication Date (Web): 07 Feb 2017 Downloaded from http://pubs.acs.org on February 8, 2017

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Discrimination Cascade Enabled Selective Detection of Single-Nucleotide Mutation Lidan Li,† Xianjin Xiao,‡ Jingyang Ge,† Manli Han,† Xu Zhou,† Lei Wang,† Xin Su,*,† and Changyuan Yu*,†

†

College of Life Science and Technology, Beijing University of Chemical Technology, Beijing,

China, 100029. ‡

Family Planning Research Institute/Center of Reproductive Medicine, Tongji Medical College,

Huazhong University of Science and Technology, Wuhan, Hubei 430030, China

*

Corresponding author

Email: [email protected]; [email protected]

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Abstract Owing to the significance of single nucleotide mutation (SNM) for personalized medicine, the detection of SNM with high accuracy has recently attracted considerable interest. Here, we present a kinetic method for selective detection of SNM based on a discrimination cascade constructed by combining the toehold strand displacement (TSD) and endonuclease IV (Endo IV) catalyzed hydrolysis. The single-nucleotide specificity of the two DNA reactions allows highly selective detection of all types of single nucleotide changes (including single-nucleotide insertion and deletion), achieving high discrimination factor with a median of 491 which is comparable with recently reported methods. For the first time, the enzyme assisted nucleic acid assay was characterized by single molecule analysis on total internal reflection fluorescence microscope (TIRFM) suggesting that the two steps do not work independently and the rate of TSD can be tuned by Endo IV facilitated conformation selection. The effective discrimination of the point mutation of BRAF gene in cancer and normal cell line suggests that this method can be a prominent post-PCR genotyping assay.

Keywords Single nucleotide mutation; Toehold strand displacement; Endonuclease IV; Single molecule analysis; Cancer diagnosis.


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Single nucleotide mutation (SNM) in human genome has proven connected with various diseases.1-3 As a result, SNM has been regarded as prominent biomarker for noninvasive early diagnosis and targeted therapy.4,5 A variety of strategies have been developed for precise SNM detection such as next generation sequencing,6 dynamic DNA nanotechnology,7,8 nanoparticle biosensors,9 electrochemical methods,10,11 synthetic nucleotide analogues,12 and enzymatic approaches.13,14 These strategies demonstrate remarkable recognition ability for single nucleotide changes. Most of them rely on single discrimination step in which SNM and wild type exhibit differential reactivity. For hybridization based probes, the discrimination step is dependent on the differential hybridization thermodynamics. However, the hybridization thermodynamics or free energy is typically close for perfectly matched and single mismatched duplex at room temperature or physiological temperature.15 Newly developed probe-pair strategies greatly improved the performance of hybridization based probes and achieved high single-nucleotide selectivity.16,17 The success of these methods suggests that multiple steps or multiple recognition probes can be helpful to eliminate the cross-reactivity and false positive readout. Toehold strand displacement (TSD) known as the core reaction of dynamic DNA nanotechnology has been used as a powerful tool to construct DNA nanostructure, DNA molecular circuits, and DNA logic gates in a programmable way.18,19 In a typical TSD reaction, the binding of invader strand on toehold domain facilitates the branch migration to release protector strand. Several researchers have demonstrated that TSD can be used for the detection of SNM by introducing mismatch in toehold domain or strand exchange domain.20,21 For example, Liang and co-workers utilized TSD reaction on chip surface achieving high selectivity for single base change.22 Meanwhile, well-understood interaction of nucleic acid and enzymes such as ligases, polymerases, endonucleases, and helicases has advanced the development of enzyme assisted nucleic acid biosensors.23 In these biosensors, high sensitivity can be readily achieved by enzyme triggered target recycling. By taking advantages of the selectivity of some enzymes, SNM detection methods have been established.14,24,25 Unfortunately, there still lack the combination of discrimination steps and the mechanism insight of the integrating nucleic acid reactions. In this regard, rational design and insightful characterization of SNM sensors with multiple discrimination steps are urgently needed.


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Single-molecule fluorescence has emerged as a powerful tool for studying the structure and dynamics of biomolecules.26,27 In vitro detection of target objects at single molecule level can be readily achieved inside a flow channel with total internal reflection fluorescence microscopy (TIRFM). By taking the advantages of transient DNA binding and single-molecule fluorescence, Walter and co-workers developed a kinetic fingerprinting approach on TIRFM for amplificationfree detection of miRNA biomarkers in serum with high sensitivity and specificity.28 Here, we describe a novel kinetic method for highly selective detection of SNM by combining TSD and endonuclease IV (Endo IV) catalyzed hydrolysis. Unlike the extensively used SNM sensing strategies relying on single discrimination step, both of the reactions utilized in this method exhibit SNM discrimination capability. High discrimination factor was therefore achieved. More importantly, the integrated reaction was characterized by the kinetics analysis at single molecule level on TIRFM suggesting TSD can be selectively accelerated by external stimuli (Endo IV). With this method, the BRAF V600E (c.1799T>A) mutation can be detected in cancer cell line. Experimental Section Materials. Endonuclease IV, Taq DNA polymerase, lambda exonuclease and ThermoPol reaction buffer were obtained from NEB (Beverly, MA). All of the oligonucleotides used in this work were synthesized and by Sangon Co. (Shanghai, China) and their sequences are listed in Table S1. All modified oligonucleotides were purified by HPLC, while unmodified oligonucleotides were purified by PAGE. (3-Aminopropyl) triethoxysilane (APTES), 3,4dihydroxybenzoate (PCA), protocatechuate dioxygenase (PCD) and Trolox were from SigmaAldrich (St. Louis, MO). mPEG-succinimidyl valerate (mPEG-SVA, MW, 5000), and biotinPEG-succinimidyl valerate (biotin-PEG-SVA, MW, 5000) were purchased from SeeBio Co. (Shanghai, China). All chemicals were used as received without additional purification. DNase/RNase free deionized water from Tiangen Biotech Co. (Beijing, China) was used in all experiments. Typical discrimination cascade assay for synthetic oligonucleotides in homogeneous solution. All reactions in homogeneous solution were carried out in 0.2 mL sealed PCR tube. 100 nM target strand and corresponding protector strand were annealed in ThermoPol buffer (20


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mM Tris-HCl, 10 mM (NH4)2SO4, 10 mM KCl, 2 mM MgSO4 and 0.1% Triton X-100, pH 8.8). Next, the invader strand and Endo IV were added simultaneously to reach final concentrations of 100 nM and 20 U/mL (65 pM), then fluorescence was recorded immediately in the FAM channel of a real-time PCR cycler (Mastercycler realplex, Eppendorf, Germany) at 37 °C with a time interval of 8 s. Reaction products (non-labeled strands) were characterized by a denaturing, 8 M urea, 20% (w/v) polyacrylamide gel. The gel was stained with SYBR gold (Life Technologies) and imaged on a gel transilluminator (ThermoFisher Scientific). Discrimination cascade assay on single-molecule platform. Single-molecule imaging surfaces (quartz slide and cover slide) were coated with a 10:1 mixture of mPEG and biotin-PEG prior to construction of the fluidic sample cell. Fluidic sample cells were constructed using two pieces of double-sided tape sandwiched between a quartz slide and glass coverslip as previously described.27 The slide surface was briefly incubated with 200 µL T50 buffer (50 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA, pH 8.0) followed by 0.5 mg/ml streptavidin for 10 min. Then, excess streptavidin was flushed out using T50 buffer. Next, 50 pM of Cy5 labeled protector strand and biotinylated TSD template strand were annealed in 1×ThermoPol buffer, and then added to the channel and incubated for 10 min. The excess was flushed out by 1×ThermoPol buffer for three times. 50 pM of FAM labeled invader strand and 5 U/mL of Endo IV solution in oxygen scavenging system (2.5 mM PCA, 25 nM PCD, 1 mM Trolox)29 contained Thermopol buffer were introduced into the channel. Prism-TIRF was used to excite FAM and Cy5 on DNA with 491 nm and 640 nm laser light. The fluorescence emission light from FAM and Cy5 was collected by an oil-immersion objective lens (APON 60XOTIRFM, 1.49NA, Olympus). The fluorescence emission light was further separated with a dichroic mirror and was detected by an EMCCD camera (IXon 897, Andor, EM gain 1000). Image acquisition was performed at a rate of 10 Hz. Genomic DNA extraction and detection of BRAF V600E c.1799T>A in cell lines. Mutant and wild type DNAs were extracted from A375 cells and HEK-293T cell by QIAamp DNA Micro Kit (QIAGEN, Germany), respectively. PCR was performed in 1×ThermoPol Buffer containing 240 nM primers (reverse primer is 5’-phosphorylated), 0.2 mM dNTPs, 0.025 U/µL Taq DNA polymerase and 0.02 ng/µL extracted DNA. The PCR amplification was carried out on GeneAmp 9700 thermal cycler (ABI, USA) for 30 cycles with a program of 94 °C denaturation 5 ACS Paragon Plus Environment

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for 30 s, 55 °C annealing for 30 s, 72 °C extension for 30 s. Next, lambda exonuclease (final concentration, 0.1 U/µL) was added to digest the 5’-phosphorylated strands which was extended by the 5’-phosphorylated primer for 20 min at 37 °C followed by inactivation of enzyme at 85 °C for 10 min. The obtained single stranded DNAs were diluted to 100 nM for discrimination

cascade assay. Results and Discussion The Principle of highly selective detection of single nucleotide mutation. Figure 1A depicts the discrimination cascade constructed by two DNA reactions. Target single strand DNA （black）is hybridized with the protector strand (red). TSD occurs upon the addition of fluorophore and quencher dually labeled invader strand (green) which contains tetrahydrofuran as a synthetic abasic site for Endo IV recognition. After TSD, Endo IV recognizes the abasic site in the invader-target duplex and cleaves the phosphodiester bond,30 resulting in the release of fluorophore labeled short strand. To achieve high single-nucleotide selectivity, we introduced two mismatches in protector-mutant duplex but single mismatch in protector-wild type duplex. On the contrast, the invader strand forms one mismatch with mutant but two mismatches with wild type. The interested mutation allele is highlighted (blue circle) in Figure 1B. This design allows differential rate of wild type and mutant in TSD step.22 As the previous work, Endo IV discriminates between doubly and singly mismatched duplexes more efficiently than a singly mismatched and fully complementary duplex.31 Therefore, the mismatch common to both wild type and mutant is introduced to enhance the Endo IV mediated discrimination. Upon the removal of the protector, Endo IV cuts the abasic site in the mutantinvader duplex containing single mismatch more rapidly than that in the wild type-invader duplex containing two mismatches. To illustrate the above process clearly, the purposefully introduced mismatch is indicated in Figure 1B. Taking advantages of two DNA reactions, discrimination cascade was readily constructed. We used a 30-nt single-stranded DNA (100 nM) as a proof of concept. As shown in Figure 2A and B, both of the TSD step and Endo IV cleavage step exhibit single-nucleotide discrimination ability yielding distinct fluorescence response for mutant and wild type. Note that in the assay of TSD alone, the fluorophore and quencher were labeled on protector and target, respectively (inset, Figure 2B). Discrimination factor (DF) was defined as the fluorescence response rate 6 ACS Paragon Plus Environment

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ratio of mutant and wild type. The response rate was calculated from the first 50 s linear portion of the time curve. By coupling the two DNA reactions, the DF was significantly enhanced compared with any single reaction and achieved within 5 min (Figure 2C and D). The reaction products were tested on 20% denaturing polyacrylamide gel electrophoresis (D-PAGE). As shown in Figure S1, invader strand (20 nt) was not found in presence of mutant, indicating its rapid degradation rate. Single Molecule Analysis of the Discrimination Cascade. To gain deep insight of the reaction mechanism of the discrimination cascade, we carried out single molecule fluorescence analysis on TIRFM. The target strand was immobilized on the PEGylated slide through biotin-streptavidin interaction. Cy5 labeled protector is pre-hybridized with target strand. The fluorescence was recorded upon the addition of fluorescein (FAM) labeled invader strand and Endo IV simultaneously. The base complementarity configuration was the same with the assay in bulk solution. Due to the excitation geometry of TIRFM, only fluorophore close to the interface can be “lightened up” (Figure 3A). TSD allows the switch of protector strand and invader strand, resulting in the fluorescence decrease of Cy5 and the enhancement of FAM. The FAM fluorescence finally decreases due to the cleavage of invader strand by Endo IV. Figure 3B shows representative single-molecule time traces obtained from a single DNA target. The fluorescence was monitored for 150 s to avoid photobleaching caused by continuous excitation. The dwell time of Cy5 fluorescence for mutant is shorter than that for wild type, suggesting more rapid protector-invader exchange when mutant is the template of TSD. The fluorescence of FAM finally decreases owing to the strand hydrolysis by Endo IV. We denoted that the period for the co-existence of the two fluorophores as strand exchange period, the period for FAM alone as enzyme degradation period, and the period for no fluorescence as reaction completion. In fact, the period of no fluorescence was rarely found in the assay of wild type during the acquisition time (bottom, Figure 3B) because of the slow rate of TSD and enzyme reaction. It is noteworthy that the re-hybridization of protector with both of the targets was not found upon the cleavage of invader strand because the rest part of invader strand blocks its binding site. The distinct kinetics signature of wild type and mutant verified the proposed mechanism. To the best of our knowledge, this is the first time for characterizing the process of enzyme assisted nucleic acid assay at single molecule level.


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TSD can be triggered by external stimuli.32,33 We hypothesized that the TSD in this method is affected by the following Endo IV cleavage step. The rate of strand exchange was compared in presence of Endo IV with that in absence of Endo IV by using the mutant strand. The distributions of the on-state lifetime of Cy5 which is labeled on protector strand in presence and absence of Endo IV are shown in Figure 4A. The dwell time is relatively shorter with Endo IV than that without Endo IV. As a consequence, the TSD in this system is accelerated by Endo IV. The acceleration of TSD was also found in bulk solution. Interestingly, the TSD in assay for wild type target was not tuned by Endo IV probably because of the large energy barrier of strand displacement (Figure 4B). The mechanisms of molecular recognition in general are classified into two classes induced-fit (IF) and conformational selection (CS) which are referred to as “binding first” and “folding first” processes, respectively.34,35 In IF, ligand binding to an unfolded conformation promotes folding, whereas in CS, the ligand selects high-affinity, folded-like structures from an ensemble and thus shifts the conformational equilibrium toward them. In our system, Endo IV serves as a ligand to promote the conformation change (branch migration in TSD). Endo IV recognizes abasic sites by flipping both the abasic site and its partner nucleotide out of the double strand DNA and shows low binding affinity towards single strand DNA.36 Thus, the mechanism of the acceleration of TSD is speculated as CS, that is, Endo IV selects TSD products and thus facilitates the branch migration. For the TSD of wild type, the interaction of Endo IV and TSD products is insufficient to mitigate the energy barrier of the mismatch limited TSD. This provides a new way to tune the rate of DNA dynamic assemble. Optimization of the reaction conditions for highly selective detection of all types of SNMs To achieve highly selective SNM detection, the assay condition was optimized. The kinetic of two DNA reactions rely on the number of complementary bases DNA structure, temperature and ionic strength. First, the length of protector strand was optimized. As shown in Figure S2A, 16nt protector offers the highest DF for C>T mutation. Next, the influence of temperature on the DF was also investigated (Figure S2B). Our system poses robustness in a broad range of temperature (25-37 °C ). The working temperature (37 °C ) of Endo IV exhibits the highest DF. High DF was also achieved at room temperature which is attractive for point of care (POC)


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diagnosis. Higher temperature was not tested because it weakens the binding of protector strand and reduces the enzyme activity. The influence of ionic strength is speculated more complicated than strand length and temperature in this system. The kinetics of DNA hybridization and enzyme activity can both be tuned by divalent and monovalent cation. To maintain the high activity of enzyme, the concentration of divalent cation (e.g. Mg2+) was not adjusted due to the divalent metal dependence of Endo IV. NaCl concentration was therefore optimized according to the DF for all types of SNMs. The DFs of all types of single nucleotide changes include insertion and deletion are shown in Figure 5A and fluorescence responses are shown in Figure S3. The corresponding concentration of NaCl used are noted. High single-nucleotide selectivity was achieved in all types of mutations. Due to the merits of the synergistic effect of the two DNA reactions, absolute discrimination was found in some types of mutations such as A>C, C>A, G>X and insertion. We speculate that this exceptionally high discrimination for C>X and G>X mutation can bet attributed to a large energy barrier with the replacement of a C-G pair with a mismatch in the assay of wild type. In summary, our system provides remarkable DF ranging from 57 to absolute discrimination with a median of 491, which is comparable with some recently reported methods.16,37,38 Detection of Single Nucleotide Mutation at Low Abundance and Mutation Detection in Cancer Cell Line. To demonstrate our method can be used for the detection of SNM at low abundance, we tested the model mutant at different fractions by using the model target. As shown in Figure 5B, the mutant can be clearly differentiated from the wild type at an abundance as low as 0.5%. BRAF mutations are associated with various types of cancer39. Detection of BRAF mutation state is of great significance for diagnosis and prognosis. BRAF V600E (c.1799T>A) mutation in A375 cancer cell line was chosen as target for demonstrating the feasibility for biological samples. Mutant and wild type targets of BRAF gene are from A375 and HEK-293T cell line, respectively. Genomic DNA were extracted from the cells, PCR reactions were carried out to quickly amplify the DNAs by using a 5’-phosphorylated reverse primer. The single strand DNAs for the discrimination cascade assay were generated by using lambda exonuclease digesting the extended phosphorylated strand. As shown in Figure 5C, the BRAF gene from cancer and 9 ACS Paragon Plus Environment

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normal cell lines exhibit distinct fluorescence response. The mutation detection at low abundance was not studied by using PCR products because the unexpected error can be introduced during the amplification step. High-fidelity polymerases are needed for post-PCR analysis. Conclusion In summary, a novel method has been developed for the selective detection of SNM. By taking the advantages of the combination of TSD and Endo IV catalyzed hydrolysis, we are able to detect all types of SNMs with high selectivity. SNM at low abundance down to 0.5% can be detected. The sensitivity can be further enhanced by coupling with microfluidic devices or other fluorescent nanomaterials with better brightness. The reaction mechanism was verified by single molecule fluorescence analysis. Both of single molecule analysis and fluorescence measurement in bulk solution suggest that the branch migration in TSD can be accelerated by Endo IV through conformational selection pathway. This holds great potential for tuning the DNA assemble rate and would find broad applications in the fields of DNA structure and dynamic nanotechnology. In combination with PCR, BRAF V600E (c.1799T>A) mutation can be detected from A375 cancer cell lines whereas the corresponding wild type from normal cell lines shows negligible fluorescence response. As a consequence, owing to the merits mentioned above, this approach would find broad applications in designing DNA nanotechnology based biosensors and POC diagnosis. Reference 1 2 3 4 5 6

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Chen, F.; Zhao, Y.; Fan, C. H.; Zhao, Y. X. Mismatch Extension of DNA Polymerases and High-Accuracy Single Nucleotide Polymorphism Diagnostics by Gold NanoparticleImproved Isothermal Amplification. Anal. Chem. 2015, 87, 8718-8723. Li, N.; Yang, Y.; He, K. M.; Zhang, F. Y.; Zhao, L. B.; Zhou, W.; Yuan, J. H.; Liang, W.; Fang, X. H. Single-Molecule Imaging Reveals the Activation Dynamics of Intracellular Protein Smad3 on Cell Membrane. Sci. Rep. 2016, 6, 33469. Blanco, M. R.; Martin, J. S.; Kahlscheuer, M. L.; Krishnan, R.; Abelson, J.; Laederach, A.; Walter, N. G. Single Molecule Cluster Analysis dissects splicing pathway conformational dynamics. Nat. Methods 2015, 12, 1077-1084. Johnson-Buck, A.; Su, X.; Giraldez, M. D.; Zhao, M. P.; Tewari, M.; Walter, N. G. Kinetic fingerprinting to identify and count single nucleic acids. Nat. Biotechnol. 2015, 33, 730-732. Aitken, C. E.; Marshall, R. A.; Puglisi, J. D. An oxygen scavenging system for improvement of dye stability in single-molecule fluorescence experiments. Biophys. J. 2008, 94, 1826-1835. Hosfield, D. J.; Guan, Y.; Haas, B. J.; Cunningham, R. P.; Tainer, J. A. Structure of the DNA repair enzyme endonuclease IV and its DNA complex: double-nucleotide flipping at abasic sites and three-metal-ion catalysis. Cell 1999, 98, 397-408. Xiao, X.; Liu, Y.; Zhao, M. Endonuclease IV discriminates mismatches next to the apurinic/apyrimidinic site in DNA strands: constructing DNA sensing platforms with extremely high selectivity. Chem. Commun.2013, 49, 2819-2821. Tang, W.; Wang, H. M.; Wang, D. Z.; Zhao, Y.; Li, N.; Liu, F. DNA Tetraplexes-Based Toehold Activation for Controllable DNA Strand Displacement Reactions. J. Am. Chem. Soc. 2013, 135, 13628-13631. Amodio, A.; Zhao, B.; Porchetta, A.; Idili, A.; Castronovo, M.; Fan, C. H.; Ricci, F. Rational Design of pH-Controlled DNA Strand Displacement. J. Am. Chem. Soc. 2014, 136, 16469-16472. Suddala, K. C.; Wang, J. R.; Hou, Q.; Water, N. G. Mg2+ Shifts Ligand-Mediated Folding of a Riboswitch from Induced-Fit to Conformational Selection. J. Am. Chem. Soc. 2015, 137, 14075-14083. Hammes, G. G.; Chang, Y. C.; Oas, T. G. Conformational selection or induced fit: A flux description of reaction mechanism. Proc. Natl. Acad. Sci. USA 2009, 106, 13737-13741. Daley, J. M.; Zakaria, C.; Ramotar, D. The endonuclease IV family of apurinic/apyrimidinic endonucleases. Mutat. Res. Rev. Mutat. 2010, 705, 217-227. Wei, B. M.; Zhang, T. C.; Ou, X. W.; Li, X. C.; Lou, X. D.; Xia, F. StereochemistryGuided DNA Probe for Single Nucleotide Polymorphisms Analysis. ACS Appl. Mater. Inter. 2016, 8, 15911-15916. Liu, W. P.; Zhu, M. J.; Liu, H. X.; Wei, J. T.; Zhou, X. M.; Xing, D. Invading stacking primer: A trigger for high-efficiency isothermal amplification reaction with superior selectivity for detecting microRNA variants. Biosens. Bioelectron. 2016, 81, 309-316. Cantwell-Dorris, E. R.; O'Leary, J. J.; Sheils, O. M. BRAFV600E: implications for carcinogenesis and molecular therapy. Mol. Cancer Ther. 2011, 10, 385-394.


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ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China (31600687, 81273631, 31400915, 21405045, 21522502), Fundamental Research Funds for the Central Universities (12060070031, 12060090029 and ys12060026025), and Health and family planning commission of Hubei province (J2017Q017). ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the website. The probe sequences used in this work are listed in Table S1. Supporting figures are included: PAGE gel analysis of discrimination cascade products, optimization of probes for model target and fluorescence response curve of all types of mutations. AUTHOR INFORMATION Corresponding Authors Email: [email protected], [email protected] Notes. The authors declare no competing financial interest.


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FIGURES AND CAPTIONS

Figure 1. Schematic representation of the discrimination cascade assay. (A) The protector strand (red) and target strand (black) are pre-hybridized. The TSD is initiated by the addition of invader strand (green). The TSD product is cleaved by Endo IV to release the FAM labeled segment resulting fluorescence enhancement. (B) The sequence of model target (C>T mutation). The interested mutation allele is highlighted by blue circle. X represents the abasic site. The purposefully introduced mismatch for enhancing Endo IV mediated single-nucleotide discrimination is indicated.


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Figure 2. (A-C) Fluorescence response of mutant and wild type in Endo IV alone assay, TSD alone assay and discrimination cascade assay within 300 s. Inset of panel B: the label scheme of TSD alone assay. (D) The comparison of DF of the three assays. Compared with the assays based on single discrimination step, discrimination cascade assay significantly enhanced singlenucleotide selectivity. The concentrations of all strands are 100 nM and Endo IV is 20 U/mL (65 pM).


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Figure 3. (A) Schematic representation of single molecule fluorescence analysis. The target strand is immobilized on imaging surface via the biotin-streptavidin interaction. Due to the excitation configuration of TIRFM, only fluorophore bound on the surface can be illuminated. TSD leads the exchange of Cy5 and FAM labeled strands. The cleavage of invader strand results in the release of FAM segment from surface. (B) Single molecule fluorescence trajectory for mutant and wild type. FAM and Cy5 are excited by 491 and 640 nm laser and detected with a time resolution of 100 ms. The concentrations of all DNA strands are 50 pM. Endo IV is 5 U/mL (16 pM). The sequences of oligonucleotide for single molecule assay are listed in Table S1.


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Figure 4. (A) single molecule toehold strand displacement in the presence (dark cyan) and in the absence (orange) of Endo IV. The addition of Endo IV shifts the distribution of the Cy5 lifetime. (B) Fluorescence response rate of TSD in homogeneous solution. The TSD in the assay of mutant is accelerated by Endo IV.


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Figure 5. (A) Summary of the DFs of all types of single nucleotide changes. Absolute discrimination is referred to the assays where wild types does not show observable fluorescence response. The assay condition for the mutation of T>C, G>C, A>C, insertion, and deletion is 1×ThermoPol buffer containing 100 mM NaCl, whereas the assay condition for others is 1×ThermoPol buffer. (B) Detection of SNM of the model target at different fractions. The total concentration of DNA targets is fixed at 1 µM. (C) Fluorescence responses of BRAF segments amplified from A375 (mutant) and HEK-293T (wild type) cell lines. The concentration of the protector strand for BRAF mutation is 400 nM. The assay condition is 1×ThermoPol buffer containing 100 mM NaCl.


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