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Improving the Signal-to-Background Ratio during Catalytic Hairpin Assembly through Both-End Blocked DNAzyme Li Deng, Yuanheng Wu, Shuxia Xu, Yurong Tang, Xinfeng Zhang, and Peng Wu ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.8b00243 • Publication Date (Web): 01 Jun 2018 Downloaded from http://pubs.acs.org on June 1, 2018

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Improving the Signal-to-Background Ratio during Catalytic Hairpin Assembly through Both-End Blocked DNAzyme *

Li Deng,† Yuanheng Wu,† Shuxia Xu,† Yurong Tang, † Xinfeng Zhang,†, Peng Wu‡, †

*

College of Materials and Chemistry & Chemical Engineering, Chengdu University of

Technology, Chengdu 610059, China ‡

Analytical & Testing Center, Sichuan University, 29 Wangjiang Road, Chengdu

610064, China *Corresponding authors: [email protected]; [email protected]

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ABSTRACT Catalyzed hairpin assembly (CHA) technique is an important DNA engineering tool for a variety of applications such as DNA nanotechnology and biosensing. Here we report a hairpin-type of both-end blocked DNAzyme to improve the signal to background ratio during CHA process. In the design, the DNAzyme activity can be blocked efficiently via locking the both ends of G-riched DNAzyme sequence in the loop and stem (blocking efficiency=96%), and easily recovered during the CHA process (activation efficiency = 94%). The both-end blocked DNAzyme is by far the most sensitive optical detection mode for monitoring the CHA process that can be used for determination of 0.05 fmol miRNA-21. The fabricated CHA-DNAzyme sensing system was also able to discriminate miRNA-21 from single/three-base mismatch miRNA-21. The feasibility of real application was also tested via detecting miRNA-21 levels in tumour cell samples. Therefore, the sensing system with the advantages of convenience, high sensitivity and selectivity, is an appealing strategy for miRNA detection. Keywords: DNAzyme; catalyzed hairpin assembly; Chemiluminescence; miRNA; Tumor cell

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Since its introduction by Pierce and his colleagues in 2008,1 catalytic hairpin assembly (CHA) has been evoked into an important DNA molecular engineering tool in recent years.2-9 Benefiting from the unique features of DNA, the CHA processes can be highly predictable and precisely controlled. CHA has been tailored to adapt to a variety of analytical applications, such as multiple assays,10 the spatial organization of biomolecules,2 and exponential signal amplification.11-13 Featuring isothermal reaction and enzyme-free, the CHA signal amplification technology exhibits competitive advantages to the classical PCR.14 Therefore, CHA provides an appealing tool for clinical diagnosis, environmental monitoring, bacterial virus detection, and etc. Aiming to boost the applications of CHA techniques, researchers have developed a variety signal read-out modes including AuNPs aggregation,15 förster resonance energy

transfer

between

fluorophore

and

organic/nanomaterial

quencher,16

electrochemiluminescent detection via terpyridyl ruthenium,17 etc. Especially, G-quadruplex DNAzymes that can efficiently catalyse the CL or coloration reaction between luminol18-20 /chromogenic substrates21, 22 and the oxidant H2O2, have been demonstrated to be a good signal-amplification reporter for chemiluminescent (CL) or visual biosensing. Since the formation or dissociation of DNAzyme can be easily triggered by analytes, it was appealing for label-free biosensing.23,

24

In previous

blocked DNAzymes, the functional structure often contains a part of free G-rich sequence for efficient target-induced formation of the G-quadruplex structure25-28 (Fig. 1A). However, the single-end blocked DNAzyme functional sequence can also partially form intermolecular DNAzyme,29 which may cause a high background signal and also prevent the subsequent CHA process (Fig. 1A). Of course, one can use fully

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blocked DNAzyme,30 but the subsequent target-induced activation efficiency is expected to be decreased, which is not benefit for the biosensing. To maintain the target-induced activation efficiency was well as lower the feasibility of the formation of intermolecular G-quadruplex, here we propose a hairpin-type both-end blocked DNAzyme to improve the S/B ratio during CHA process. Since the sequence of DNAzyme is locked at its both ends in the hairpin struture, the formation of intermolecular G-quadruplex is largely restricted; also the enzymatic activity of blocked DNAzyme was easily recovered during the CHA process that triggered by miRNA. Using this strategy, a sesnsing system with high S/B ratio was fabricated for ultrasensitive and lablel-free detection of low aboundant miRNA in biological samples.

EXPERIMENTAL SECTION Materials. All the chemical reagents were at least analytical grade. The used nucleic acids in this study (Table S1) were synthesized by Sangon Biotech, and diethyl pyrophosphate (DEPC) and solid phase RNase-Be-Gone were purchased from the same company. Luminol and hemin were bought from Sigma-Aldrich. Other chemical reagent such as chloroform, ethanol anhydrous, triton X-100, H2O2 etc. were provided by Kelong Reagent Co. (Chengdu, China). RNase-free tip and centrifuge tube purchased from the Thermo Fisher scientific life technologies Shanghai Trade Co., Ltd. The equipment that cannot be soaked is wiped three times with solid phase RNase-Be-Gone. 18.25 MΩ cm-1of ultrapure water is used for all experimental processes.

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Preparation of probes. The sequence of H1 and H2 (Table S1) were designed according to the principle of CHA. To form a complete hairpin structure, both of probe H1 and probe H2 were denaturized at 95 °C for 5 min and cooled gradually at a dropping speed about 5 oC/min until to the room temperature (25 oC). The prepared probes are then stocked at 4 °C for further use. Procedure of sensing miRNA-21. Different concentration of miRNA standard solutions/sample solution were added to 500 µL of the mixed solution of 4 nM H1 and 3 nM H2 that was prepared in pH 7.4 buffer solution consisting of 25 mM HEPES, 25 mM KCl, 200 mM NaCl, 150 mM NH4Cl and 0.05% triton X-100 and 1% DMSO. Subsequently, the solution was incubated at 33 °C for 1 h. Then hemin was introduced into the system with its final concentration of 6 nM, and the solution was incubated for half an hour. 100 µL of the above solution was added to the mixture solution of luminol (0.5 mM) and H2O2 (5mM). The CL spectra were recorded by an F-280 fluorescence spectrometer (Gangdong Sci. & Tech. Development Co., Ltd., Tianjin). The CL signal was recorded by an ultra-weak chemiluminescence detector (RLF-1A, Xi’an Remex Analytic Instruments Co., China). The blocking and activation efficiencies are calculated according to equation (I) and (II), respectively.

E =

E =

( ) ( ) ( )

× 100%

( ) ( ) ( ) ( )

(Ⅰ)

× 100%

(Ⅱ)

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Where E and E correspond to blocking efficiency and activation efficeincy, respectively ;and I( ) , I(!") , and I(#$%& ")

stand for CL intensities of G-rich

hemin aptamer, H2, and miRNA-21 adding to the system.

Polyacrylamide Gel Electrophoresis. The verification experiment of target-catalyzed activation

of

both-end

blocked

DNAzyme

system

was

demonstrated

by

polyacrylamide gel electrophoresis. The samples was loaded on 10% native polyacrylamide gel (acrylamide: bis-acrylamide= 29:1) and run in 1× TBE (Tris-borate-EDTA) buffer at room temperature for 50 min (running voltage, 120 V), then dyed with ethidium bromide (EB) and recorded using a Bio-Rad digital imaging system. Total RNA extraction from different cells. Normal human liver cell line (LO2), human cervical cancer cell lines (Hela), and human hepatocellular liver carcinoma cell line (HepG2) are processed with cell counting. Then, RNAs were extracted from the cell lines using the total-RNA extraction kit. LO2 were used as the normal miRNA expression level control. The total RNA extraction from cell was conducted according to the total RNA extraction kit instruction.

RESULTS AND DISCUSSIONS Design of hairpin-type both-end blocked DNAzyme for CHA. The structure of the both-end blocked DNAzyme was shown in Fig. 1A. One end of the DNAzyme was fixed in the loop part of the hairpin, and the other end was fully blocked in the stem. In this design, the formation of intermolecular G-quadruplex is largely restricted

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without target miRNA. Meanwhile, the target-induced dissociation can be occurred from the loop part without obstruction. Moreover, the hairpin-type both-end blocked DNAzyme can be easily integrated into the subsequent CHA process for enzyme- and label-free amplification.

Figure 1 (A) scheme of single/both-end blocked DNAzymes; (B) schematic illustration of the catalytic hairpin assembly triggered by miRNA-21 with both-end blocked H2; and (C) signal to background ratios of the two blocked DNAzymes. Experiment conditions: miRNA-21,500 pM; hemin, 6 nM; H1, 4 nM; H2, 3 nM; H2O2, 5 mM; luminol, 0.5 mM.

The scheme for CHA-based cycling is shown in Fig. 1B and Fig. S1. To facilitate the CHA process (Fig. 1B), the hairpin-type both-end blocked DNAzyme (H2) and another hairpin DNA containing recognition region for the target (H1) were employed as the fuel. Firstly, the target (here exemplified as miRNA-21) triggered hybridization reaction between the probe H1 and miRNA, resulting in opening up the hairpin structure and formation of the H1-target complex. Further hybridization of H2 with the H1-target complex leads to free of the G-rich functional region for subsequent formation of the G-quadruplex DNAzyme, catalysing the oxidation of luminol for chemiluminescence readout. Meanwhile, the target is also released for next cycle and

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activation of the DNAzyme. Through the above cycling, ultrahigh sensitivity and low background were achieved. To illustrate the advantage of the both-end block DNAzyme in CHA-based amplification, a single-end blocked DNAzyme was designed as a control.31 The background signal from the single-end blocked DNAzyme is much higher than that of the both-end blocked DNAzyme (Fig.1C). This is probably ascribed to the formation of intermolecular G-quadruplex in single-end blocked DNAzyme. Here, such formation was confirmed using ThT (a good G-quadruplex structure indicator32) as the probe (Fig. S2). More importantly, the formation of intermolecular G-quadruplex can prevent the opening of H2 hairpin, and thus hindering the CHA process. The catalytic turnover numbers for the both-end and single-end blocked DNAzyme are 5.01 and 1.67, respectively (See the detail calculation in Fig. S3). Hence, a much higher S/B ratio was obtained for the both-end blocked DNAzyme (14.5) over the single-end blocked counterpart (1.9). Validity of CHA-activating the blocked DNAzyme. For confirming the CHA progress, the signal of 1 nM of miRNA-21 was detected by the sensing system (H1 + H2). The resulted products were analysed by gel electrophoresis. Fig. 2A shows that H1 and H2 cannot open spontaneously and form the complementary pairing (B vs. H1 and H2) in the absence of the target. In the presence of the target, a new band which was in accordance with the dsDNA formed by H1 and H2 was observed. The results of the polyacrylamide gel electrophoresis demonstrated that the target miRNA can catalyse the assembly between H1 and H2 hairpins. Subsequently, the formation of G-quadruplex DNAzyme was verified with UV-vis absorption. The binding of hemin

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to G-quadruplex DNA can be characterized by the red shift and hyperchromicity of the Soret band of hemin.33 In Fig. 2B, the red shift and hyperchromicity effect was indeed found after addition of miRNA, confirming the formation of G-quadruplex.

Figure 2 Validation of the CHA process and DNAzyme activation: (A) Gel electrophoresis of the activation products; and (B) UV-vis absorption spectra of hemin in different conditions. Nominations in (A): M, DNA markers; B, mixture of H1 and H2 (blank), dsDNA, H1 and H2 were denaturized to form dsDNA; 1-2, two paralleled activations.

Estimation of the blocking and activation efficiency. Under the optimal conditions (Fig. S4-S8), the blocking and activation efficiency were evaluated. Upon assembling with hemin, the G-rich functional sequence exhibited a high peroxidase-like activity, as revealed by the strong luminol CL signal (Fig. 3A). By blocking the both ends in the hairpin structure (H2), the blocked DNAzyme showed extremely low CL background signal, which is similar to that of hemin only. The blocking efficiency is estimated to be ~96%. After addition of H1 hairpin probe, negligible CL increase was observed (Fig. 3B), demonstrating that H1 probe cannot open the hairpin structure of H2. However, upon addition of 0.5 nM miRNA to the mixture of H1 and H2, sharp

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peroxidase-like activity increase was observed from the corresponding luminol CL (Fig. 3B), indicating that the blocked DNAzyme could be activated via the target-induced CHA. Such activation efficiency is estimated to be as high as 94%, which is benefiting from the high catalytic turnover number using both-end blocked DNAzyme.

Figure 3 Estimation of the blocking and activation efficiency: (A) the CL spectra of luminol before and after blocking of DNAzyme; and (B) the CL spectra of luminol before and after activation of DNAzyme by CHA process. Experiment conditions: hemin, 6 nM; H1, 4 nM; H2, 3 nM; H2O2, 5 mM; luminol, 0.5 mM. The CL spectra were measured with a fluorescence spectrometer.

Analytical performance of the sensing system. We used different concentrations of miRNA-21 to assess the sensitivity of the both-end blocked DNAzyme-CHA system with a CL analyzer. Upon varying the miRNA-21 concentration varied from 0.5 pM to 2 nM, stepwisely increased CL intensity was observed. The linear responses for miRNA-21 were ranging from 0.5 pM to 500 pM (0.05 fmol to 50 fmol in amount). As can be seen from Fig. 4A, the background signal from the both-end blocked

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DNAzyme after CHA process is considerably lower than that from single-end block DNAzyme. The limit of detection (LOD, 3σ) is about 0.2 pM (0.02 fmol in absolute LOD), which is 45-fold lower than that obtained from single-end blocked strategy. The ultralow LOD is benefiting from the higher sensitivity (high catalytic turnover) and much lower background (no formation of intermolecular DNAzyme) of the proposed system. As tabulated in Table 1, the proposed both-end blocked DNAzyme is by far one of the most sensitive detection modes for single CHA-based sensing of miRNA. It can be also used for sensitive sensing of DNA (Fig. S9).

Figure 4 (A) miRNA-21 concentration-dependent CL intensity from the both- and single-end blocked DNAzyme-CHA system; and (B) selectivity evaluation of the both-end blocked DNAzyme-CHA system for miRNA-21 sensing. The CL signal was detected by a CL analyzer.

The selectivity of the proposed sensing system for miRNA-21 detection is evaluated against several other miRNAs. Fig. 4B indicate that adding other miRNAs including single/three-based mismatched miRNA yielded a quite low CL singal, whereas a high CL signal was found upon addition the target miRNA ( 0.1 nM of 11


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miRNA-21). This result indicates excellent selectivity of the proposed sensing system for miRNA detection. Table 1 Comparing with other detection modes for single CHA-based sensing of miRNA/DNA Strategya Both-end blocked

Detection method

Label

Dynamic-range

LOD

Ref. This

miRNA

CL

-

0.5 pM-500 pM

0.2 pM

miRNA

CL

-

25 pM-2 nM

9 pM

miRNA

Fluorescence

-

1 pM-1 nM

NM

34

miRNA

Fluorescence

TAMRA

Up to-16 nM

47 pM

35

CHA/2-AP/ThT

miRNA

Fluorescence

2-AP

0.5 nM-50 nM

72 pM

36

CHA/2-AP/MBs

miRNA

Fluorescence

2-AP

40 pM –40 nM

3.5 pM

37

CHA/AuNRs/Cy5

miRNA

Fluorescence

Cy5,-SH

0.5 nM-50 nM

NM

38

miRNA

Fluorescence

FAM,BHQ1

1 pM-2 nM

NM

39

DNA

Colorimetry

-

0.1 nM-10 nM

DNAzyme-CHA Single-end blocked DNAzyme-CHA

CHA/GO/SG

CHA/GO /TAMRA

CHA/FAM/BHQ1 CHA/GO/AuNPs/TMB a

Analyte

57.4

work This work

40

pM

Abbreviations：LOD=limit of detection; NM=not mention; GO=graphene oxide; CHA=catalyzed hairpin

assembly;

FAM=fluorescein;

2-AP=2-aminopurine;

TAMRA=

ThT=thioflavin

carboxytetramethylrhodamine; T;

MBs=molecular

AuNRs=gold

beacon;

nanorods;

Cy5=cyanine-5;

TMB=3,3,5,5-tetramethylbenzidine.

Detection of miRNA-21 expression levels in different tumor cell lines. Nowadays, miRNAs have been considered to be a new type of bio-markers. 41, 42 To evaluate the feasibility of the proposed sensing strategy for miRNA detection, tumor cell extractions were monitored. For this purpose, the cell lysates of the HepG2 and HeLa were analyzed. As shown in Fig. 5A, the cell lysate from an increasing number of 12


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HepG2 cells leads to linearly increase in CL response with a correlation coefficient (R2) of 0.9573. Additionally, the difference of miRNA-21 expression levels between human normal cells (LO2 cells) and tumor cells (HeLa and HepG2 cells) is compared. The normal cell line LO2 is set as the normal miRNA-21 expression quantity control (defined as unit 1). The cell lysate from HepG2 cells exhibits much high CL response over LO2 cells, while that of the HeLa cells is only slightly higher (Fig.5B). Therefore, miRNA-21 is over-expressed in the HepG2 cells rather than the HeLa cells. To confirm the signal is from miRNA-21 in cell lysates, 10 nM of DNA that is complementary to miRNA-21 was used as a blocker. The CL signal DNA, as shown in Fig. S10, was significantly reduced for HepG2 after adding the complementary DNA. This demonstrated that the CL signal was indeed from miRNA-21 in cell lysates. Thus, the proposed both-end blocked DNAzyme-CHA system can be employed for monitoring of miRNA expressed from cancer cells.

Figure 5 (A) Sensitivity and linearity of the HepG2 tumor cell lines (B) Comparison of miRNA-21 expression levels in different cell lines. The CL signal was detected by a CL analyzer.

CONCLUSION 13


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This work proposed a hairpin type of both-end blocked DNAzyme for significantly improving the S/B ratio of the CHA-DNAzyme sensor. The enzymatic activity of DNAzyme was efficiently blocked by locking the both-ends of the G-riched DNAzyme sequence in a hairpin structure, showing a quite low background (S/B ratio of 14.5 for both-end blocked strategy versus 1.9 for single-end block strategy). Meanwhile, the both-end blocked DNAzyme hairpin can be dissociated via target-catalysed hairpin assembly process, leading to the activation of the blocked DNAzyme for catalysed generation of the luminol CL. The both-end blocked DNAzyme is by far the most sensitive detection mode for CHA-based sensing of miRNA with an LOD as low as 0.2 pM. Also it exhibited excellent discrimination capability for distinguishing single-base mismatch miRNA. Importantly, it can be applied for detection of miRNA expression levels in cells. Hence, we believe the both-end

blocking

strategy

may

be

advantageous

for

future

design

of

DNAzyme-based biosensors.

ACKNOWLEDGEMENT The authors gratefully acknowledge the financial support from the National Natural Science Foundation of China (Nos. 21475013 and 21605010) and the Sichuan Youth Science and Technology Foundation (Grant 2016JQ0019).

ASSOCIATED CONTENT Supporting Information The supporting information is available free of charge on the ACS publication website. Supporting information includes sources: the sequences of DNA or RNA in this work;

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Total RAN extraction procedure; Specific Principle of CHA in this work; the

fluorescence response of ThT in different DNAzyme sequence; Comparison of cycle efficiency of two systems; the optimization of the H1 concentration; the optimization of the H2 concentration; the optimization of the hemin concentration; the optimization of the incubation temperature; the optimization of the incubation time; Linearity of detection of DNA by the CHA-DNAzyme Sensing system; the signal inhibition after adding complementary DNA

Notes The authors declare no competing financial interest.

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