Direct Detection of Nucleic Acid with Minimizing Background and

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Direct Detection of Nucleic Acid with Minimizing Background and Improving Sensitivity Based on a Conformation-Discriminating Indicator Lixuan Zhu, Zhihe Qing, Lina Hou, Sheng Yang, Zhen Zou, Zhong Cao, and Ronghua Yang ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.7b00349 • Publication Date (Web): 25 Jul 2017 Downloaded from http://pubs.acs.org on July 27, 2017

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Direct Detection of Nucleic Acid with Minimizing Background and Improving Sensitivity Based on a Conformation-Discriminating Indicator

Lixuan Zhu,† Zhihe Qing,*,†,‡ Lina Hou,† Sheng Yang,† Zhen Zou,† Zhong Cao,† Ronghua Yang*,†,‡

†

Hunan Provincial Key Laboratory of Materials Protection for Electric Power and

Transportation, Hunan Provincial Engineering Research Center for Food Processing of Aquatic Biotic Resources, School of Chemistry and Biological Engineering, Changsha University of Science and Technology, Changsha 410114, P. R. China ‡

State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry

and Chemical Engineering, Molecular Science and Biomedicine Laboratory, Hunan University, Changsha 410082, P. R. China. *To whom correspondence should be addressed: E-mail: [email protected] (Z. Qing); [email protected] (R. Yang). Fax: +86-731-88822523.

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ABSTRACT As well known, the nucleic acid indicator-based strategy is one of the major approaches to monitor the nucleic acid hybridization-mediated recognition events in biochemical analysis, displaying obvious advantages including simplicity, low-cost, convenience and generality. However, conventional indicators either hold strong self-fluorescence or can be lighted by both ssDNA and dsDNA, lacking absolute selectivity for a certain conformation, always with high-background interference and low sensitivity in sensing; and additional processing (e.g. nanomaterial-mediated background suppression and enzyme-catalyzed signal amplification) is generally required to improve the detection performance. In this work, a carbazole derivative, EBCB, has been synthetized and screened as a

dsDNA-specific fluorescent indicator.

Compared with conventional indicators under the same conditions, EBCB displayed a much higher selective coefficient for dsDNA, with little self-fluorescence and negligible effect from ssDNA. Based on its superior capability in DNA conformation-discriminating,

high

sensitivity

with

minimizing

background

interference was demonstrated for direct detection of nucleic acid, and monitoring nucleic acid-based circuit with good reversibity, resulting in low detection limit and high capability for discriminating base-mismatching. Thus, we expect that this highly-specific DNA conformation-discriminating indicator will hold good potential for application in biochemical sensing and molecular logic switching.

Keywords: Nucleic acid, sensitive, direct detection, fluorescent, indicator


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Due to the continued demand in environmental monitoring and disease diagnosis, the development of advanced recognition probes for functional biochemical molecule analysis is very significant.1-8 As well known, the hybridization between a nucleic acid probe and its complement target is one of the most efficacious biomolecular recognition events in biosensing,9-13 and the signal transduction among with the recognition events is always the focus in the design and application of nucleic acid probes.14-17 Fluorescent nucleic acid indicator is one of the major approaches to monitor the nucleic acid hybridization-mediated recognition events in biochemical analysis, displaying obvious advantages including simplicity, low-cost, substantivity and generality.18-24 However, on one hand, strong self-fluorescence of indicators leads to failure in constructing “turn-on” strategy for in situ detection without isolating the indicator-target hybrids from an excess of the unbound indicators (type I in Figure 1). On the other hand, although some indicators can be lighted by nucleic acids from low to high fluorescence, they can be enhanced by both ssDNA and dsDNA, lacking absolute selectivity for a certain conformation, always with high-background and false-positive interference in sensing (type II in Figure 1). Additional processing (e.g. nanomaterial-mediated background

suppression and enzyme-catalyzed signal

amplification) was generally required to improve the detection performance for the type I and II.25-30 Therefore, it is challenging and significative to find a specific DNA conformation-discriminating fluorescent indicator, with little self-fluorescence, target-triggered “turn-on” signal, and even high sensitivity with minimizing


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background interference (type III in Figure 1). In this work, inspired by the challenge and significance, the interactions between a series of fluorescent indicators and DNAs were investigated. As a result, a carbazole derivative,

ethyl-4-[3,6-bis(1-methyl-4-vinylpyridium

iodine)-9H-carbazol-9-yl)]

butanoate (EBCB), was synthetized and screened as a highly dsDNA-specific fluorescent indicator, with little self-fluorescence and negligible effect from ssDNA, leading to high sensitivity with minimizing background interference in DNA conformation transformation-mediated sensing. Compared with other conventional indicators, EBCB displayed a much higher selective coefficient (φ) for dsDNA (Figure 1), which resulted in great capability for direct detection of nucleic acid. A higher sensitivity was demonstrated in discriminating base-mismatching trough EBCB, and this effective indicator was successfully exploited to monitor nucleic acid-based circuit, with good reversibility.

EXPERIMENTAL SECTION Chemicals and Apparatus. All DNA sequences used in this work were purchased in Sangon Biotech Co., Ltd. (Shanghai), the detailed sequence information was listed in Table S1. The fluorescence indicator, ethyl-4-[3,6-Bis(1-methyl-4-vinylpyridium iodine)-9H-carbazol-9-yl)] butanoate (EBCB) was synthesized following our previous reported method (Figure S1).31 Conventional nucleic acid indicators including gold view (GV), SYBR gold (SG), SYBR green I (SGI), and ethidium bromide (EB), were commercially purchaseed from Dingguo Biotechnology CO., Ltd (Beijing, China). Exonucleases III (Exo III) was purchased from Fermentas Co., Ltd. (America).


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3-(N-morpholino) propanesulfonic acid (MOPS) and other inorganic salts of at least analytical grade were abtained from Sinopharm Chemical Reagent Co., Ltd. (China). All buffer solutions were prepared using ultrapure water which was produced by a Millipore purification system (18.2 MQ resistivity). All pH measurements were carried out by a model 868 pH meter (Orion). Agarose gel electrophoresis was carried out in an electrophoresis tank (Liuyi, Beijing, China), and the electrophoresis results were recorded by a gel imaging system (ChemiDoc XRS+, Bio-RAD). Ultraviolet-visible (UV-vis) light absorption spectra were recorded on a UV-2600 UV-vis spectrometer (Shimadzu, Japan). The fluorescence spectra were carried on fluorospectrophotometer systems (ASOC-10 and TCM-1000, Photo Technology International, Birmingham, NJ, USA). Screening of the Conformation-Specific Indicator. In order to investigate the conformation-discriminating ability of fluorescent indicators, single stranded DNA (ssDNA) and double stranded DNA (dsDNA) were used to interact with various fluorescence dyes, respectively, including EBCB, GV, SG, SGI and EB. Fluorescent assay was performed in 10 mM MOPS buffer containing 150 mM NaCl (pH 7.2). 500 nM ssDNA or 250 nM dsDNA was added into 500 µL MOPS buffer containing 500 nM EBCB. Similarly, conventional nucleic acid indicators were also investigated respectively, according to the method mentioned above. Their fluorescence spectra were recorded on PTI ASOC-10 Fluorescence System, with their maximum excitation wavelength, 470 nm for EBCB, 490 nm for SG, 495 nm for SGI, 495 nm for GV, 500 nm for EB.


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Electrophoresis Characterization of Conformation-Specificity To verify the much higher specificity of EBCB in discriminating DNA conformation, compared with conventional nucleic acid indicators, electrophoresis characterization was carried out as a visual manner: DNA samples were prepared by adding 5 µM ssDNA (DNA1) or 2.5 µM dsDNA (2.5 µM DNA1+2.5 µM DNA2) in 30 µL of buffer solutions; 5 µL of each DNA sample was then mixed with 1 µL EBCB (100 µM), and incubated for 5 min, followed by the addition of 1 µL 6×loading buffer; 5 µL of the resulted mixture was analyzed using gel electrophoresis in 3% (w/w) agarose, and ran in 0.5×TBE buffer at 80 V for 5 minutes; The resulted gel was finally visualized using an imaging system (ChemiDoc XRS+, Bio-RAD). For SG and SGI, the electrophoresis process is the same as that for EBCB. For GV and EB, the dyes were previously mixed in the gel as noted in their instructions. Investigation of Interaction Mode between EBCB and dsDNA. To study the interaction mode between EBCB and double-stranded DNA, different concentrations of dsDNA (0, 50, 200, 500 and 1000 nM) were respectively added to 500 µL of MOPS buffer solution (pH 7.2) in the presence of EBCB (2 µM), and incubated for 2 min. The UV-vis absorption spectra of each mixture were measured in the wavelength range of 330-550 nm. Direct Detection of Nucleic Acid and Base-Mismatching Recognition. In the fluorescence experiments for DNA target detection, the response of EBVB/ssDNA1 and molecular beacon (MB) to different concentration of target DNA (DNA 2) was characterized. For the detection of DNA target in EBCB/DNA1 system, DNA 2 of


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different concentrations (0, 2, 5, 10, 20, 40, 60, 80, 100, 120, 150 nM) were added into 500 µL MOPS buffer containing 300 nM EBVB and 150 nM DNA1, and then incubated at room temperature for 2 min, the fluorescence spectra were collected from 500 to 700 nm with 470 nm excitation. For the MB detection strategy, DNA 2 of different concentrations (0, 0.5, 1, 2, 5, 10, 20, 30, 40, 60, 80, 100 nM) were added into 500 µL MOPS buffer containing 150 nM MB, and then incubated at room temperature for 1 h, the fluorescence spectra were collected from 500 to 700 nm with 490 nm excitation. In base-mismatching recognition study, 250 nM DNA 1 or MB in 500 µL MOPS buffer was used; and 250 nM base-mismatched DNAs were detected by EBCB/DNA1 system and MB respectively; the detection processes were the same as that mentioned above. Monitoring Reversible Conformation Transformation. To monitor the DNA conformation transformation between single-stranded and double-stranded forms，and construct a tunable molecular-lamp between off and on states by using EBCB as a highly-efficient indicator, a 3′ terminus-recessed complementary DNA (DNA 7) and Exo III were used to alternately turn the fluorescence on/off. Detailedly, 500 nM EBVB and 250 nM DNA1 were added into a 1× Exo III buffer of 500 µL, containing 100 mM NaCl, to form the EBCB/DNA1 system; 250 nM DNA7 was introduced into the above system for the in situ formation of dsDNA, and the fluorescence signal was recorded. Subsequently, Exo III of 200 U was added into the above mixture and incubated for 20 min to allow the enzyme-catalyzed digestion reaction of DNA 7, and the fluorescence signal was recorded again. Alternately, DNA 7 and Exo III were


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alternately used to regulate the fluorescence for some times.

RESULTS AND DISCUSSION Screening of the Conformation-Specific Indicator. Inspired by the challenge and significance, we aimed to synthetize and screen a highly-specific dsDNA-lighted fluorescent

indicator.

First

of

ethyl-4-[3,6-bis(1-methyl-4-vinylpyridium

all,

a

carbazole

iodine)-9H-carbazol-9-yl)]

derivative, butanoate

(EBCB), was synthetized according to our previous method (Figure S1), and different small molecular dyes including EBCB and several conventional indicators were used to interact with DNAs. The sequences of all DNAs used in this work are listed in Table S1. As shown in Figure S2, conventional indicators either had strong self-fluorescence in the absence of any DNA (e.g. Goldiew, GV), or could respond to both ssDNA and dsDNA (e.g. SYBR Gold, SG), lacking absolute selectivity in DNA conformation–discriminating, and resulting in low sensitivity with high-background interference in DNA conformation transformation-mediated sensing. Attractively, EBCB could be only lighted by dsDNA, with little self-fluorescence and negligible effect from ssDNA (Figure 2A). The fluorescence responses of different indicators to ssDNA and dsDNA are summarized in Figure 2B by increasing signal-to-background ratio, (FDNA-FI)/FI, where FI is the fluorescence intensity of each indicator itself, and FDNA is that after the addition of ssDNA or dsDNA. When EBCB was used as the signal indicator, a specific phenomenon to dsDNA was observed: almost no signal increasement occured as a function of the addition of ssDNA, whereas a much higher increasement in signal-to-background ratio was displayed for dsDNA, implying a


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high sensitivity with minimizing background signal for direct detection of a target DNA. To further illustrate conformation specificity toward dsDNA compared with ssDNA, selectivity coefficient (φ) was introduced and defined as the following formula: φ = (FdsDNA−FI) /(FssDNA−FI),32 where the concentration of dsDNA was a half of that of ssDNA, ensuring that the total amount of nucleotides was

same. From

Figure 2C, compared with other conventional indicators, EBCB displayed a prominent selectivity coefficient (φ) toward dsDNA . In addition, It was easy to find that EB and SGI could also discriminate dsDNA from ssDNA in a degree (Fiure S2C and D). To measure the effect of DNA sequences on EB, SGI and EBCB, other two groups of DNA were also investigated (Figure S3 and S4). Although, for EB and SGI, the level of interference from different ssDNAs was discrepant, there was a much smaller selectivity coefficient than EBCB in each group. The conformation specificity was also visually demonstrated by gel electrophoresis (Figure 2D), one could see that conventional indicators could be lighted by both ssDNA and dsDNA, despite a little difference in brightness; only EBCB could high-specificly recognize dsDNA with no interference from ssDNA. Interaction Mode between EBCB and dsDNA. Subsequently, the interaction mode between EBCB and dsDNA was investigated. As shown in Figure 3A, with the increasing of dsDNA concentration, the UV-Vis absorption intensity of EBCB decreased gradually, with redshift of absorption spectra. According to the theory verified by previous reports,33,34 the hypochromism and redshift stem from the


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intercalating action of the dye in dsDNA, with an electronic interaction between the intercalator and DNA bases. In addition, the effect from the electrostatic interaction between EBCB and the negatively charged phosphate backbone of DNA was also investigated by titrating sodium chloride (NaCl) in the buffer. From Figure 3B, one could see that there was a little enhancement of EBCB fluorescence when NaCl of low concentration (5 mM) was added into the work buffer; this was due to the fact that cation can facilitate the hybridization of two complementary strands, favoring the impaction of EBCB in dsDNA. When the concentration of NaCl increased unceasingly (to 60 mM), the EBCB fluorescence almost remained constant, which indicated that the electrostatic interaction between EBCB and the negatively charged phosphate backbone of DNA does not have obvious effect on the dsDNA-induced enhancement of EBCB fluorescence. In addition, to investigate the preference of EBCB in binding with DNA stretches, four 20-mer DNA homopolymers (A20, T20, C20, G20) were designed to carried out the fluorescence enhancement experiment. As shown in Figure S5, all single-stranded homopolymers had no effect on the fluorescence signal of EBCB; attractively, a significant enhancement was observed when in the presence of both A20 and T20, while negligible enhancement was observed when in the presence of both C20 and G20. Thus, we could conclude that this indicator has a preference for bingding with A-T stretches. Direct Detection of Nucleic Acid. Due to its high selectivity and sensitivity in DNA conformation-discriminating, EBCB was used as an efficient and label-free


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strategy for direct detection of nucleic acid. The principle was based on the fact that the complementary target could hybridize with the probe ssDNA to form dsDNA, along with the intercalating action of EBCB and its significant fluorescence enhancement. As shown in Figure 4, DNA 1 was used as a model probe, and DNA 2 was detected as a model target. EBCB fluorescence increased gradually with the increasing of target concentration from 0 to 150 nM (Figure 4A). The relationship between fluorescence intensity of EBCB and target concentration was plotted in Figure 4B. Expectedly, good detection performance for nucleic acid detection was demonstrated by EBCB, there was a wide linear range from 2 to 100 nM (inset in Figure 4B), with a detection limit of 1.75 nM determined by 3σ. Visually, the titration of the target nucleic acid was also carried out by gel electrophoresis (Figure 4C), with the increasing of the target concentration, the electrophoresis bands became brighter and brighter. For further evaluation of the EBCB-based strategy, a hairpin molecular beacon (MB), which has been commonly used as a standardized tool for detection of nucleic acids,35-38 was designed to detect the same model target (DNA 2). The MB was labeled with 6-carboxy fluorescein (FAM) as a fluorescent reporter at its 5’ end, and 4-(4-dimethylamino phenylazo) benzoic acid (DABCYL) as a fluorescent quencher at its 3’ end, and the loop of MB contained the same sequence as DNA 1. When the MB hybridized with the target, its conformation transformed from hairpin to open state, resulting in fluorescence recovery. The fluorescence of MB increased gradually with the increasing of target concentration (Figure S6A), a good linear range from 1 to100


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nM and a detection limit of 1.22 nM (by 3σ) were obtained by MB (Figure S6B). Thus, it was not difficult for us to evaluate that EBCB, as well as MB, holds an analogous sensitivity for nucleic acid detection, but without complicated design and modification. Base-Mismatching

Recognition.

Besides

sensitivity,

the

recognition

of

base-mismatching is another important criterion for evaluating a new strategy in nucleic acid detection. The capability of EBCB for distinguishing base-mismatch was investigated. As shown in Figure 5A, DNAs with different base-mismatching number were characterized through EBCB fluorescence. Attractively, compared with the fully-complementary target (red curve), an obvious signal decreasement could be observed when only a mismatching base existed in the detected sequence (green curve); and with the increasing of base-mismatching number, EBCB fluorescence decreased significantly. For comparison, the same sequences were also detected by the MB (Figure 5B) and other indicators (Figure S7). The results were summarized in Figure 5C, one could find that EBCB had molecular beacon-like capability for recognition of base-mismatching, and much higher sensitivity than other conventional indicators. In addition, this capability was also visually demonstrated by gel electrophoresis (Figure 5D), with the increasing of base-mismatching number in the detected sequence, the bands of DNA/EBCB dimminged and disappeared posteriorly (Figure 5D, up), which was analogous to the MB detection strategy (Figure 5D, down).


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Monitoring Nucleic Acid-based Circuit. Inspired by its highly-specific DNA conformation-discriminating capability, EBCB was also exploited to construct a tunable molecular circuit between off and on states (Figure 6A). Detailedly, with the addition of the 3′ terminus-recessed complementary DNA (cDNA) (DNA 7) into the EBCB/DNA 1 system, DNA 7 hybridized with DNA 1 to form dsDNAs, and the intercalating action of EBCB in the dsDNAs’ grooves turned on the fluorescence of EBCB. To turn off its fluorescence, exonuclease III (Exo III) which can gradually remove mononucleotides from a blunt or recessed 3′ terminus of dsDNA was used to digest DNA 7, resulting in the transformation from dsDNA to ssDNA, EBCB was released and its fluorescence was turned off. As designed, the fluorescence of EBCB was alternately regulated by cDNA and Exo III (Figure 6B and C), with good reversibity. The fluorescence switching was also visually verified by gel electrophoresis (Figure 6D). These results demonstrated that EBCB as a conformation-specific indicator can dynamically monitor the nucleic acid-based circuit, displaying good potential in construction of optical molecular logic, with excellent reversibility.

CONCLUSIONS In summary, a carbazole derivative, EBCB, has been successfully demonstrated as a highly-specific dsDNA-lighted fluorescent indicator, through the intercalating action in dsDNA grooves. Compared with conventional indicators under the same conditions, EBCB displayed a much higher selective coefficient for dsDNA, with little self-fluorescence and negligible effect from ssDNA. Based on its superior capability


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in DNA conformation-discriminating, EBCB displayed great potential for biochemical applications, such as direct detection of nucleic acid and monitoring nucleic acid-based circuit. Results demonstrated that the performance of EBCB-based strategy was much better than that of other conventional indicators, with minimizing background and improving sensitivity. Some further studies on the application of EBCB in molecular logic switching and biochemical sensing are currently being undertaken in our lab.

ASSOCIATED CONTENT Supporting Information Supporting Information Available: The following files are available free of charge. Detailed sequence information of oligonucleotides used in this study, synthetic route of EBCB, spectra of fluorescent indicators in the presence of ssDNA or dsDNA, detection of the model target DNA by MB, recognition of base-mismatching by different fluorescent indicators.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] (Z. Qing). * E-mail: [email protected] (R. Yang). Fax: +86-731-88822523. Notes The authors declare no competing financial interest.


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ACKNOWLEDGMENT We are grateful for the financial support through the National Natural Science Foundation of China (21605008, 21575018, 21505006), the Hunan Provincial Natural Science Foundation (2016JJ3001), the Scientific Research Fund of Hunan Provincial Education Department (16C0032, 16C0033), the Open Fund of State Key Laboratory of Chemo/Biosensing and Chemometrics of Hunan University (2015003) and Hunan Provincial Engineering Research Center for Food Processing of Aquatic Biotic Resources (2016GCZX04).

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ACS Sensors

Figures and Captions: indicator indicator indicator +ssDNA +dsDNA (FssDNA) (FdsDNA) (FI)

conformation selectivity

30

type I

no selectivity

type II

low selectivity

type III

high selectivity

√

20

10

0 GV

SG

SGI

EB

EBCB

selectivity coefficient ((FdsDNA-FI)/ (FssDNA-FI))

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

Page 22 of 28

Figure 1. The general types of nucleic acid indicators. EBCB has been synthetized and found as a highly-specific dsDNA-lighted fluorescent indicator (type III), with a much higher selective coefficient (φ) compared with conventional indicators.


Page 23 of 28

(B)

40

EBCB + dsDNA20

1.0

ssDNA20

EBCB + ssDNA20

0.8

dsDNA20

EBCB

(FDNA-FI) / FI

Normalized fluorescence

(A)

0.6 0.4 0.2

30 20 10

0.0 0 500

550

600

650

700

750

GV SG SGI EB EBCB

Wavelength / nm

(D) GV

SG

SG I

EB

EBCB

30 20 +dsDNA20

+ssDNA20 +dsDNA20

+dsDNA20

+ssDNA20

+ssDNA20 +dsDNA20

0

+ssDNA20

10

+dsDNA20

(FdsDNA-FI)/(FssDNA-FI)

(C) 40

+ssDNA20

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

ACS Sensors

GV SG SGI EB EBCB

Figure 2. (A) The fluorescence spectra of EBCB under different conditions in MOPS buffer (10 mM MOPS, 150 mM NaCl, pH 7.2); (B) The fluorescence responses of different

indicators

to

ssDNA20

and

dsDNA20,

through

increasing

signal-to-background ratio, (FDNA-FI)/FI, where FI was the fluorescence intensity of each indicator itself, and FDNA was that after the addition of ssDNA or dsDNA; (C) The selectivity coefficient (φ) of each indicator for dsDNA compared with ssDNA, φ=(FdsDNA−FI)

/(FssDNA−FI);

(D)

Electrophoresis

characterization

of

DNA

conformation specificity. Here used ssDNA20 was DNA 1; dsDNA20 was the mixture of DNA 1 and DNA 2, whose sequence information is detailedly listed in the Table S1. The concentration of dsDNA (250 nM) was a half of that of ssDNA (500 nM), ensuring that the total amount of nucleotides was

same. The concentration of

indicators was 500 nM.


ACS Sensors


1.2

1.0

0

1.0

.8 1 µM

.6 .4 .2 0.0 350

400

450

500

Wavelength / nm

550

.8 .6

Normalize fluorescence

(B)

(A) Normalized absorbance

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

Page 24 of 28

1.0 .5 0.0 0 10 20 30 40 50 60 [Na+] / mM

.4 .2

0.0 500 550 600 650 700 750 800 Wavelength / nm

Figure 3. (A) The UV-Vis absorption spectra of EBCB (2 µM) in the presence of dsDNA20 of different concentrations, in MOPS buffer (10 mM MOPS, 150 mM NaCl, pH 7.2); (B) The fluorescence spectra of EBCB/dsDNA20 with titrating sodium chloride in the work buffer. Inset was the plot of fluorescence intensity vs Na+ concentration.


Page 25 of 28

(A)

(B)

1.2

8

150 nM

1.0 6

[target DNA]

.8 .6

F/F0


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

ACS Sensors

0

6

4

y=0.0543x+1.4804 2 R =0.9877

4

.4 2

.2 0.0 500

2 0

0 550

600

650

700

Wavelength / nm

0

30

60

20 40 60 80 100

90

120 150

¡[[target DNA] / nM

(C)

0

[target DNA] / nM

150

Figure 4. (A) Spectra of EBCB/DNA 1 in the presence of target DNA of different concentrations, in MOPS buffer (10 mM MOPS, 150 mM NaCl, pH 7.2). EBCB fluorescence increases gradually with the increasing of target concentration from 0 to 150 nM. (B) The relationship between the fluorescence intensity of EBCB and target concentration. Inset in B shows the linear range from 2 to 100 nM; (C) Gel electrophoresis characterization of the titration of the target nucleic acid, the electrophoresis bands became brighter and brighter with the increasing of the target concentration.


ACS Sensors

(A)

(B) 1.2 6: 1+DNA2 (mis 0) 5: 1+DNA3 (mis 1) 4: 1+DNA4 (mis 2) 3: 1+DNA5 (mis 4) 2: 1+DNA6 (random) 1: EBCB/DNA1

1.0 0.8 0.6 0.4 0.2

0.0 500 550 600 650 700 750 800 Wavelength / nm

(C) 1.2 1.0 0.8



1.2

(FdsDNA-Fmis) / FdsDNA

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

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6: 1+DNA2 (mis 0) 5: 1+DNA3 (mis 1) 4: 1+DNA4 (mis 2) 3: 1+DNA5 (mis 4) 2: 1+DNA6 (random) 1: MB

1.0 0.8 0.6 0.4 0.2 0.0 500

550

600

650

Wavelength / nm

(D)

SG GV EB SGI EBCB MB

EBCB

a

0.6

b

c

d

e

0.4 0.2 MB

0.0 s1 s2 s4 om lank mi mi mi and b r

Figure 5. Spectra of EBCB/DNA 1 (A) and MB (B) in the presence of detected DNAs with different base-mismatching number, in MOPS buffer (10 mM MOPS, 150 mM NaCl, pH 7.2). The loop of MB contained the same sequence as DNA 1; (C) Recognition of base-mismatching by different indicators and MB, through fluorescence change, (FdsDNA-Fmis)/ FdsDNA, where FdsDNA was the fluorescence intensity in the presence of the full-complementary target, and Fmis was that in the presence of the corresponding base-mismatching target ； (D) Electrophoresis demonstration of the capability for discriminating base-mismatching. The number of base-mismatching from a to d was 0, 1, 2 and 4, e is the result of the detection of random sequence. With the increasing of base-mismatching number in the detected sequence, the bands of DNA/EBCB dimminged and disappeared posteriorly (up), which was analogous to the MB detection strategy (down).


Page 27 of 28

(A)

(B)

= EBCB

= =DNA 1

0.6 0.4 0.2 0.0 500

=DNA 7

Exo III

Exo III

0.8 cDNA

cDNA

1.0 (n circles)

ON


OFF

550

600

650

700

Wavelength / nm

(D)

(C) Normalized fluorescence

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

ACS Sensors

ON

1.0 0.8

=

0.6 0.4

1 2 3 4 5 6 7 8

0.2 OFF

0.0 0

2

4

6

8

10

12

Circles

Figure 6. (A) Schematic representation for the construction of a reversible molecular circuit with the capability of switching between off and on states; (B) The fluorescence spectra of EBCB circularly regulated by cDNA (DNA 7) and Exo III; (C) The switching of the molecular-lamp between two states; (D) Visual demonstration of fluorescence switching by gel electrophoresis. The bands with addition of cDNA were signed with odd number, and that with succedent addition of Exo III were signed with even number.


ACS Sensors

Graphic for TOC only indicator indicator indicator +ssDNA +dsDNA (FssDNA) (FdsDNA) (FI)

conformation selectivity

30

type I

no selectivity

type II

low selectivity

type III

high selectivity

√

20

10

0 GV

SG

SGI

EB

EBCB


selectivity coefficient ((FdsDNA-FI)/ (FssDNA-FI))

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

Page 28 of 28

Direct Detection of Nucleic Acid with Minimizing Background and

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