Palindromic Molecule Beacon-Based Cascade Amplification for

Feb 7, 2018 - Although only one type of probe is involved, the system can execute triple and continuous polymerization strand displacement amplificati...
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Palindromic molecule beacon-based cascade amplification for colorimetric detection of cancer genes Zhifa Shen, Feng Li, Yifan Jiang, Chang Chen, Huo Xu, Congcong Li, Zhe Yang, and Zai-Sheng Wu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04895 • Publication Date (Web): 07 Feb 2018 Downloaded from http://pubs.acs.org on February 8, 2018

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Palindromic molecule beacon-based cascade amplification for colorimetric detection of cancer genes Zhi-Fa Shen,a,b# Feng Li,a,b# Yi-Fan Jiang,b Chang Chen,b Huo Xu,b Cong-Cong Li,b Zhe Yangb and Zai-Sheng Wu*b

a

Henan key laboratory of immunology and targeted drugs, Research Center for

Molecular Oncology and Functional Nucleic Acids, Henan Collaborative Innovation Center of Molecular Diagnosis and Laboratory Medicine, School of Laboratory Medicine, Xinxiang Medical University, Xinxiang, Henan 453003, PR China b

Cancer Metastasis Alert and Prevention Center, Fujian Provincial Key Laboratory of

Cancer

Metastasis

Chemoprevention

and

Chemotherapy,

Pharmaceutical

Photocatalysis of State Key Laboratory of Photocatalysis on Energy and Environment, College of Chemistry, Fuzhou University, Fuzhou, 350002, China. #

The two authors contributed equally to this work.

*Corresponding Author: Email: [email protected] (Z.S. Wu)

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Abstract A highly sensitive and selective colorimetric assay based on a multifunctional molecular beacon with palindromic tail (PMB) was proposed for the detection of target p53 gene. The PMB probe can serve as recognition element, primer, polymerization template and contains a nicking site and a C-rich region complementary to a DNAzyme. In the presence of target DNA, the hairpin of PMB is opened, and the released palindromic tails inter-molecularly hybridize with each other, triggering the autonomous polymerization/nicking/displacement cycles. Although only one type of probe is involved, the system can execute triple and continuous polymerization strand displacement amplifications, generating large amounts of G-quadruplex fragments. These G-rich fragments can bind to hemin and form the DNAzymes that possess the catalytic activity similar to horseradish peroxidase, catalyzing the oxidation of ABTS by H2O2 and producing the colorimetric signal. Utilizing the newly-proposed sensing system, target DNA can be detected down to 10 pM with a linear response range from 10 pM to 200 nM, and mutant target DNAs are able to be distinguished even by the naked eye. The desirable detection sensitivity, high specificity and operation convenience without any separation step and chemical modification demonstrate that the palindromic molecular beacon holds the potential for detecting and monitoring a variety of nucleic acid-related biomarkers.

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Introduction Cancer is responsible for one in eight deaths worldwide, which arises as a result of changes that have occurred in the DNA sequence of the genomes of cancer cells.1 Over the past quarter of a century, many important genes responsible for cancers have been discovered and their mutations were precisely identified, and the pathways through which they act were characterized.2,3 This provides a new tool for the diagnosis of cancers at a stage when they are still can be curable by conventional surgical or medical methods.2 Though less dramatic than cures, prevention and early diagnosis are perhaps the most promising and effective way to fight cancer.4 Thus, robust evaluation of cancer-related genes is of great significance. In the proof-of-concept experiments, the tumor suppressor gene p53 was often chosen as the target model because it is the most frequent target for abnormalities in every type of cancer.5 For genetic studies or molecular diagnosis, the sequencing method provides reliable large-scale DNA sequencing but has the disadvantages of target pre-amplification, chemical modification, high operational cost and/or a long turnaround time depending on the size and complexity of the gene.6-8 Thus, this method is impractical for clinic diagnosis. Polymerase chain reaction (PCR) is the most widely-used amplification technique, which relies on thermal cycling and Taq DNA polymerase for primer-directed target amplification. Due to its relatively simple reaction and operation, excellent sensitivity, PCR become a cost-effective choice for laboratories.9 However, the intrinsic drawback is that PCR needs precise thermal

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cycling between several temperatures, and the thermal cycler itself is too expensive for low-resource setting area. As an alternative of PCR, isothermal amplification replicates nucleic acid at a single temperature, which greatly favors point-of-care (POC) diagnosis.10 The well-established isothermal amplification methods have been reviewed by some excellent reviews10-13, which include helicase-dependent amplification (HAD), strand displacement amplification (SDA), loop-medicated isothermal amplification (LAMP), rolling circle amplification (RCA), recombinase polymerase amplification (RPA), nucleic acid sequence-based amplification (NASBA), and so on. However, most of these amplification strategies amplify nucleic acid templates using one or more primers, whereas others utilize a functional template as primers.13 The involved primers usually induce the non-specific amplification or false-positives due to the design oversight or accidental collision.14-16 Therefore, the development of primer-free isothermal amplification that is simpler and more sensitive is still required. A palindrome reads exactly the same from the 5’ end to the 3’ end on both strands of DNA.17 They have been implicated in various DNA-mediated processes including gene expression18, transcription17, and chromosomal translocations19 and so on. Palindromic sequence has the unique properties of hybridization between each other and naturally, has the potential of serving as primers. As a result of bypassing the primer, the inherent side reaction of the primer and beacon is avoided to reduce the background signal and improve the detection sensitivity.20 Constructing an efficient transducer accompanied by a further signal

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amplification is needed to improve the performance of sensing system.21,22 Colorimetric assays are preferred for the commercialization purpose duo to its simple interpretation and easy handling, which is based on the common AuNP moiety23,24, HRP-catalyzed conversion of 3, 3’, 5, 5’-tetramethylbenzidine (TMB) or 2, 2’-azino-bis (3-ethylbenzothiazoline)-6-sulfonate disodium salt (ABTS) to the colored product25-27, conversion of luminol–iodophenol–H2O2 to its colored product through alkaline phosphatase, etc.21 An interesting DNAzyme that is frequently used for biosensing is a biocatalytic nucleic acid with peroxidase mimicking catalytic activity. For this molecule, binding to hemin yields a G-quadruplex structure that can catalyze the oxidation of ABTS by H2O2 to the colored product ABTS.+, making the development of simple, practical, low cost and rapid colorimetric method possible.28,29 Nicking endonucleases are special restriction enzymes that cleave only one DNA strand on a specific site of double-stranded DNA substrate.30,31 The cleavage peculiarity, coupled with polymerase, has been utilized to develop SDA-based signal amplification strategies14,27,32-34, but several nucleic acid probes are often needed to be designed. In the newly-proposed sensing system, one molecular beacon with palindromic tail at 3’ end (PMB) was proposed for the colorimetric assay DNA targets without any chemical modification and additional nucleic acid probes. Integration of SDA technique into a HRP mimicking DNAzyme amplification is able to boost the performance of the colorimetric sensor. In the presence of target, the PMB was opened and the locked palindromic tail by the stem-loop structure was released. In

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this case, the palindromic fragments at the 3’ end of PMBs can hybridize with each other and the polymerization displacement by polymerase occurs. As a result, the target was peeled off and recycled. The polymerization product can be cleaved by the nickase at the designed recognition site, generating a new site for the initiation of replication and displacement. Akin to target gene, the replaced nicked fragments (NF) can hybridize with new PMBs and initiate the next round of reactions, leading to the unique palindromic strand displacement amplification (P-SDA). On the other hand, the NFs can form G-quardruplex DNAzymes in the presence of hemins and catalyze the oxidation of ABTS by H2O2, producing the highly sensitive colorimetric signal. The design of PMB, working mechanism and assay performance are elucidated in the text. Experimental section Materials and reagents All oligonucleotide sequences designed in this research were synthesized by Invitrogen; Thermo Fisher Scientific, Inc., (Shanghai, China), and the detailed sequences are listed in Table S1. All oligonucleotides were dissolved in TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0) and stored at 4 ℃ refrigerator before usage. The Klenow fragment (KF) polymerase (3’-5’ exo-), the restriction endonucleases Nt.BbvCI and the Low Molecular Weight DNA Ladder were purchased from New England Biolabs (Beijing, China). Hemin and 2, 2’-azino-bis (3-ethylbenzothiazoline)-6-sulfonate disodium salt (ABTS) were purchased from Sigma-Aldrich (Shanghai, China), while the deoxynucleotide triphosphates (dNTPs),

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SYBR Green I and H2O2 were supplied by Dingguo Changsheng Biotechnology Co., Ltd., (Beijing, China). Fetal bovine serum (FBS, HyClone) was stored at -20 ℃ and was defreezed at room temperature before use. All other chemicals were of analytical grade without further purification. Ultrapure water used to prepare all solutions was obtained through a Kerton lab MINI water purification system (Changsha, China) (resistance > 18 MΩ cm). Polyacrylamide gel electrophoresis (PAGE) The 12% non-denaturing polyacrylamide was freshly prepared in laboratory and the electrophoresis was performed in 0.5×TBE buffer (44.5 mM Tris-boric acid, 1 mM EDTA) at a constant voltage of 80 V. The SYBR Green I was used as the fluorescent indicator. The stained gel was scanned by a chemiDox XRS Imaging system with the image acquisition and analysis software Image Lab (Bio-RAD, USA). Target detection The following procedure was used to detect the target p53 gene: 2.5 µL of 10× NEBuffer 2 (500 mM NaCl, 100 mM Tris-HCl, 100 mM MgCl2, 10 mM DTT, pH 7.9 at 25 ℃), 0.5 µL of 10 µM PMB, 0.5 µL of 5 U/µL Klenow fragment (3’-5’ exo-), 0.5 µL of 10 U/µL Nt.BbvCI nickase, 1 µL of 10 mM dNTPs and 20 µL of target DNA at specific concentration are successively added into an 0.5-ml Eppendorf tube, and the final volume of reaction solution is 25 µL. After mixing gently, the mixture was incubated at 37 ℃ for 90 min. Subsequently, the reaction was terminated by heat inactivation at 80 ℃ for 20 min, and then the resulting solution was cooled to room temperature. Subsequently, 2 µL of 50 µM hemin, 65 µL of 2× HEPES buffer (50 mM

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HEPES, 0.4 M NaCl, 40 mM KCl, 2% DMSO, 0.1% Triton X-100, pH= 5.2) and 33.5 µL of ddH2O were added and incubated for 30 min at room temperature, allowing the DNA properly fold into G-quadruplex-hemin DNAzyme. Finally, 2 µL of 40 mM ABTS and 2.5 µL of freshly prepared 5 mM H2O2 were successively added and incubated for 30 min at 37 ℃. All the experiments were conducted using the standard procedure unless otherwise specified. Colorimetric measurement The resulting solution mentioned above (110 µL) was transferred to the cuvette and the absorbance spectrum was collected from 350 nm to 500 nm by a UV-2700 spectrophotometer (Shimadzu, Japan) with software of Hyper UV version 2.42. The absorbance peak at 414 nm was used to evaluate the capability of the newly-proposed sensing system. Results and discussion Design and working principle of PMB probe Compared with the traditional nucleic acid sensing system, only one type of probe, multifunctional palindromic fragment-molecule beacon (PMB), is involved in the newly-proposed sensing system. The four functional regions of designed PMB and the working principle are illustrated in Scheme 1. Region I (light green) is C-rich region that

is

complementary

to

horseradish

peroxidase

(HRP)

mimicking

hemin/G-quadruplex DNAzyme. Region II (red) is the fragment complementary to the nicking site of restriction endonuclease Nt.BbvCI. Region III (orange) is target recognition region that is the single-stranded loop part of PMB and can hybridize with

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target gene. Region IV (light blue) is a palindromic sequence. It is well known that the palindromic sequences are able to hybridize with each other between two. For the designed PMB, the hybridization between palindromic sequences was locked in initial state by the stem-loop structure. However, in the presence of target gene, upon the hybridization of PMB to target, the prelocked palindromic sequences are released and intermolecular hybridization occurs (①). Accordingly, the polymerization could be initiated by polymerase, resulting in extended PMB/extended PMB duplex (EP-duplex) (③) and releasing the target gene (②). The displaced target can bind to another PMB, achieving target-recycling (called the first SDA). Subsequently, the Nt.BbvCI specifically recognizes the newly-formed nicking site located in EP-duplex domain and cleaves the replicated one of region II (④ ). The cleavage can induce the polymerization reaction at the nicked site, generating a new complete EP-duplex (⑤) and displacing the nicked fragment (NF) (⑥) leading to the second SDA. As much as target DNA behaviors, the released nicked fragments bind to other PMBs ⑦ and open the stem-loop structures. NF/PMB duplexes can interact with each other via the hybridization between their released palindromic fragments. This promotes the polymerization reaction and displacement reaction, generating the EP-duplex (⑧) and the NF (⑨, the third SDA). Moreover, the polymerization/displacement/nicking cycle is able to proceed repeatedly, resulting in dramatic accumulation of NFs. The free NF forms the G-quadruplex DNAzyme in the presence of hemin (⑩) and catalyzes the oxidization of ABTS by H2O2 to the colored ABTS.+, generating an amplified colorimetric signal.

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To avoid the unwanted non-specific extension of PMB, two-base fragment was designed to dangle at 3’ end via protruding out from the locked palindromic sequence. In a word, the proposed PMB without any chemical modification serves as the recognition element, polymerization primer and template and is capable of executing triple strand displacement. Thus, the sensing system is expected to possess substantial advantages of low cost, rapid response, visible observation, convenient operation, and so on.

Scheme 1. Scheme for the colorimetric detection of target p53 gene using a versatile

PMB, where triple cyclical strand displacement amplification is involved. The resulting

products,

DNAzyme

units,

generated from

the

combination of

polymerase/dNTPs with nickase implement the colorimetric signal amplification via catalyzing the oxidation of ABTS by H2O2 in the presence of hemin.

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Feasibility of target gene detection Integration of so many functional elements into a single probe in a cooperative way seems a great challenge. Thus, the feasibility of this colorimetric sensing system for target p53 gene detection was initially investigated. Comparative experiments were performed and the results are shown in Figure 1. As seen from curve a, a weak UV-Vis absorption peak was observed, indicating that hemin alone almost could not possess the catalytic activity toward ABTS-H2O2. Even if adding one or more other species into the solution, there is still no change in the absorption intensity as shown in curve b, c, d and f, suggesting the several species involved in the proposed colorimetric assay system exhibit the extremely low absorption intensity. In contrast, the introduction of target gene causes a dramatic increase in UV-Vis absorption peak (curve f) while the background (curve e) was kept at a low level. Significantly, colorimetric assay result (Figure 1A inset) indicates the existence of target p53 gene can be easily detected by naked eyes without the need of any instrument. The feasibility of the colorimetric sensing system was also confirmed by native PAGE gel analysis and the result is displayed in Figure 1B. The band in lane b is PMB, while the band in lane c is the target/PMB duplex. Several bands with different molecular weight appear in lane d after the introduction of polymerase, which may be attributed to the intra-/inter-molecular polymerization products and/or intermediates.35 Complicated and similar bands appear in lane f and lane e, which should be seemingly attributed to the introduction of polymerization and nickase. But, the real reasons are still unknown and might deserve a detailed investigation in the future. Nevertheless,

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compared with lane e, a unique band with very low molecular weight marked by arrow symbol is seen in lane f. This band is named NF band because it is similar to the band in lane g consisting of only one species, NF. It is noteworthy that the brightness intensity of NF band cannot reflect the real amount of NFs because the Sybr Green I dye exhibits a very poor ability to embed into the ssDNAs compared with double-stranded DNAs (dsDNA). In this colorimetric assay system, like target gene, the NF is designed to unlock the stem via hybridizing with PMB. The NF function was explored and the detailed results are shown in Figure S1. The measured data demonstrated that the proposed PMB-based sensing system can operate efficiently for the detection of target DNA.

Figure 1. The feasibility of PMB-based colorimetric sensing system for target DNA detection. (A) UV-Vis absorption spectra of samples: (a) hemin; (b) hemin + PMB4; (c) hemin + PMB4 + target; (d) hemin + PMB4 + target + polymerase; (e) hemin + PMB4 + polymerase + nickase; (f) hemin + PMB4 + polymerase + nickase+ target. Inset: photographed images of the same samples. (B) Native-PAGE analysis of samples (b) to (f) mentioned above and the commercially synthesized nicked fragment (g). The samples were prepared using the standard procedure depicted in Experimental Section, and the concentration of PMB4, target DNA, NF, Klenow polymerase, Nt.BbvCI and dNTPs are 200 nM, 200 nM, 600 nM, 0.1 U/µL, 0.2 U/µL and 400 µM, respectively. The letter of “M” refers to the low molecular weight ladders.

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Optimization of PMB sequence and reaction time To achieve the desirable assay performance, it is essential to choose a PMB with high signal transduction capability that is dependent on several crucial factors, including the palindromic fragment, the location of nicking site and the tightness of stem. Thus, seven PMBs were designed and their detailed base sequences are seen in Table S1. All assay experiments were conducted under identical conditions, and the measured data are shown in Figure 2A. One can see that PMB4 can provide the highest colorimetric signal. If the crucial factors cannot work in a cooperative way, for example, extending palindromic fragment (PMB3), shortening the stem (PMB5), changing the nicking site position (PMB1) or replacing the palindromic fragments (PMB2 and PMB7), the optical signal could possibly be compromised. Evidently, it is important to optimize the PMB sequence before the detection of target gene. Nevertheless, the PMB optimization is simple, convenient, time and cost-effective because all experiments are accomplished in homogenous solution, only one nucleic acid probe is needed, and no any chemical modification is involved. We noted that longer enzymatic reaction time allowed the signal increase, but the absorption peak background also increased. Thus, the dependence of signal intensity on the incubation time was explored. As shown in Figure 2B, a period of 90 min was used for the enzymatic reaction in subsequent experiments because the highest signal-to-noise ratio is offered at this time point. Interestingly, target DNA can be observed by the naked eye within 20 min as shown in Figure S2.

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Figure 2. Signal intensity, A-to-A0 ratio, generated from different PMB-contained

sensing systems (PMBs1-7) (A) and at various incubation times for enzymatic reactions (B), where A and A0 are the UV–vis absorption intensities recorded in the presence and absence of target DNA, respectively. PMB4 was employed in the samples of (B). The assay experiments were carried out as depicted in Experimental Section. The concentration of target DNA is 200 nM for panel A or 150 nM for panel B. The error bars are the standard deviations of measured data based on three independent experiments at each target DNA sample.

Assay sensitivity To investigate the assay sensitivity of the proposed strategy, we collected the UV-vis absorbance spectra induced by various concentrations of target gene under the optimized conditions. As shown in Figure 3A, the absorbance peak increases monotonically with the increase of target DNA concentrations ranging from 0 to 300 nM. Compared with the blank, 10 pM target DNA is able to induce an increase in absorption peak (shown in inset I) and thus is defined as the detection limit. Excitingly, as demonstrated in Inset II, the color change induced by 5 nM target gene is visible to the naked eye.

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The Figure 3B clearly represents a good dynamic relationship between colorimetric signal and the target concentration over 5-orders of magnitude. The linear regression equation is A=0.06565C+0.1218 with the correlation coefficient (R2) of 0.9954, where the A and C represent the absorption peak value and target concentration, respectively. Compared with some previous reports15,34, the newly-proposed sensing system achieved a comparable or superior sensitivity, even if only one probe and several mixing steps were involved. More information on the comparison in terms of assay capability between the proposed signaling strategy and previous sensing methods are shown in Table S2. The excellent assay performance achieved by this simple system should be attributed to the palindromic fragment-mediated triple-SDA.

Figure 3. The capability of PMB-based system to quantify target DNA. (A) UV-vis absorption spectra in the presence of various target concentrations of target DNA ranging from 0 to 300 nM. Inset I: the spectra at the low concentration of target; Inset II: the photographic images of corresponding samples. (B) The linear relationship between colorimetric signal and target concentration and the corresponding regression equation. A, C and R2 represent the absorption peak intensity at 414 nm, target DNA concentration and correlation coefficient, respectively. The assay experiments were carried out as described in Experimental Section. The error bars represent the standard deviations of three independent experiments.

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Detection specificity Specificity is crucial for a regular medical check-ups and early screening for cancers. The p53 protein is responsible for a range of critical cellular functions, and its biological activity is often compromised by the presence of gene mutations. Mutant p53 protein is the most frequently mutated tumor suppressor in cancers.36,37 To inspect the assay specificity of the sensing system for detecting p53 gene, mutant targets possessing one, two or three point mutations (details seen in Table S1, MT1 is closely associated with the clinical cancers) were detected and the results are shown in Figure 4(A). If the signal induced by wild-type gene is defined as 100%, the corresponding relative absorbance of MT1, MT2 and MT3 are 96%, 68% and 18%, respectively. In order to improve the specificity, we shortened the matching base fragment between the target and PMB, and the signals are shown in Figure 4(B). The relative absorbance of mismatched targets decreases with reducing matching bases, and about 40% corresponding to SMT3 is achieved, indicating the proposed strategy holds the potential application in early cancer diagnosis. Additionally, although it is not easy to distinguish MT1 by naked eyes (Figure 4A Inset), SMT1 can be correctly recognized (Figure 4B Inset) even with only one-base mismatched base.

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Figure 4. The detection specificity of PMB-based colorimetric sensing system. (A)

Relative absorbance intensity induced by one-, two- or three-base mismatched targets. The relative absorbance was calculated by the formula of (A-A0)/(At-A0)×100%, while A, At and A0 represent the absorbance peak induced by mutant target DNAs, wild-type target DNA and the Blank, respectively. The relative absorbance upon the wild-type p53 gene was defined as 100%. The concentration of target DNAs involved was 200 nM. The Inset indicates the photographed images of the corresponding samples. (B) Relative absorbance induced by the shortened targets (ST2-ST4) and the corresponding single-base mismatched targets (SM2-SM4). The Inset is the photographic images of the corresponding samples. The error bars represent the standard deviations of three independent measurements. Interference study On the basis of consideration of the excellent performance of PMB-based sensing system, we explore its potential practical application by determining the target DNA in biological media. The fresh fetal bovine serum (FBS) was chosen because it contains a variety of enzymes and other complexes that can often interfere with the operation of biosensors. The measured results are shown in Figure S3. One can see the substantial difference in absorbance peak or in solution color between NEBuffer and FBS solution although the absorbance signal decreases and the background increases with increasing FBS concentration, suggesting the FBS without any pretreatment has an effect on the developed biosensor. Even so, the target gene can still be well detected by the UV-Vis absorption spectrum or the naked eyes even in 10% FBS-contained solution. Conclusion In summary, we have developed a desirable sensing system for the simple, sensitive

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and selective colorimetric detection of target gene via designing a functional palindromic MB probe (namely PMB). With the help of polymerase and nickase, the target gene can specifically promote the autonomous formation of horseradish peroxidase (HRP) mimicking hemin/G-quadruplex DNAzyme and substantial accumulation, provide the amplified colorimetric readout. This colorimetric assay displays high sensitivity with the detection limit of 10 pM due to the powerful triple SDA effect and excellent selectivity that one-base mutation could be discriminated even without any chemical modification and apparatus. Considering the peculiarity of palindromic sequence, the primer-free concept developed in this work could be extended to other detection techniques, such as hyperbranched rolling circle amplification, in future. Without engaging tedious primer design, the newly-proposed sensing system is an attractive candidate for the simple and robust nucleic acid detection platform for point-of-care diagnostics. Acknowledgments This work was supported by National Natural Science Foundation of China (NSFC) (grant NO: 21775024) and Independent Research Project of State Key Laboratory of Photocatalysis on Energy and Environment (NO. 2014CO1). Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Oligonucleotide

sequences

involved

in

this

study;

Characterization

of

NF-medicated reactions; Required incubation time to observe the target by the naked

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