Selection and Application of ssDNA Aptamers against Clenbuterol

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Article

Selection and application of ssDNA aptamers against clenbuterol hydrochloride based on ssDNA library immobilized SELEX Nuo Duan, Wenhui Gong, Shijia Wu, and Zhouping Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04951 • Publication Date (Web): 06 Feb 2017 Downloaded from http://pubs.acs.org on February 6, 2017

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Journal of Agricultural and Food Chemistry

Selection and application of ssDNA aptamers against clenbuterol hydrochloride based on ssDNA library immobilized SELEX Nuo Duan,a Wenhui Gong,b Shijia Wu,ac Zhouping Wang *ad

a

State Key Laboratory of Food Science and Technology, School of Food Science

and Technology, Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi 214122, China b c

Market Supervision and Administration Bureau, Taicang, 215400, China

School of Food and Biological Engineering, Jiangsu University, Zhenjiang 212013, China d

National Engineering Research Center of Seafood, School of Food Science and Technology, Dalian Polytechnic University, Dalian, 116034, China

1

ACS Paragon Plus Environment


1

ABSTRACT

2

Clenbuterol Hydrochloride (CLB) is often abused as additive feed for livestock

3

to decrease adipose tissue deposition and increase growth rate. It raised a potential

4

risk to human health through the animal product consume. In this study, aptamers

5

with higher affinity and specificity was screened through sixteen selection rounds

6

based on ssDNA library immobilized systematic evolution of ligands by exponential

7

enrichment (SELEX) technique. After cloning and sequencing, five aptamer

8

candidates were picked out for affinity and specificity assays based on graphene oxide

9

(GO) adsorption method. The results shown that the aptamer CLB-2 bind specifically

10

against CLB with the dissociation constant Kd value of 76.61±12.70 nM. In addition,

11

an aptamer based fluorescent bioassay was established for CLB analysis. The

12

correlation between the CLB concentration and fluorescent signal was found to be

13

linear within the range of 0.10 ng/mL to 50 ng/mL with a limit of detection of 0.07

14

ng/mL. It has been further applied for the determination of CLB in pork samples,

15

showing its great potential for sensitive analysis in food safety control.

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KEYWORDS: aptamer, clenbuterol hydrochloride, library immobilization, SELEX

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INTRODUCTION

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Clenbuterol hydrochloride (CLB), a kind of β-Agonist, is widely used as

20

bronchodilator, tocolytic and heart tonic medicine in clinic.1 Unfortunately, CLB has

21

also been used illegally as veterinary drug or additive for livestock to improve the

22

conversion rate of lean meat and animal growth rate.2,3 Due to long half-life time and

23

slow metabolism, excessive CLB accumulates in the animal and transfers into the

24

human body via animal products.4,5 People who consume food containing CLB over a

25

long period of time would probably experience headache, palpitation, muscular

26

tremors and acute poisoning.6 CLB has been forbidden to use as veterinary drug or

27

additive for animal in many countries.7 Thus, it is urgent to develop a rapid, sensitive

28

and cost-effective analysis method for CLB detection in animal products for the

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assurance of consumer healthy.

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Currently, the instrumental analysis and immunoassay are the main analytical

31

methods available for CLB detection. The instrumental analysis including high

32

performance liquid chromatography (HPLC),

33

spectrometry (LC-MS), 9 gas chromatography mass spectrometry (GC-MS)10 rely on

34

the expensive instrument, complicated sample treatment and professional personnel

35

operation. The immunoassays like enzyme-linked immunoassay (ELISA),

36

electrochemical immunoassay, 12 fluorescence immunoassay

37

specificity, accuracy and sensitivity. But the preparation of specific antibody is a

38

tedious and time-consuming process, and has animal ethic arguments. The stability of

39

antibodies is also susceptible to temperature and other environmental factors.14

8

liquid chromatography mass

13

11

have advantages of

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Aptamer is a short single-stranded DNA (ssDNA) or RNA sequences that form

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unique spatial conformation which provide the basis for excellent affinity and

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specificity toward their targets. Additional advantages of aptamers include selection in 3



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vitro, economical and facile synthesis, easy modification and stability during

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storage.15 Till now, various aptamers have been isolated by SELEX against numerous

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targets ranged from large cells to small ions.16-18 Whether the target is large cells or

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small molecules, the protocols for selection of aptamers are based on some type of

47

affinity separation of binding and unbinding sequences. When the target is large

48

bacteria or cells, the binding sequences could be separated from unbinding sequences

49

by centrifugation, which is simple and effective.16 However, when it comes to small

50

molecules that often mean chemically modified them and then attached them to the

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solid-state matrix like magnetic beads or sepharose. Gu et al reported an aptamer

52

targeted to diclofenac selected by Mag-SELEX.19 Diclofenac was covalent attached to

53

magnetic beads by carboxyl and amine groups. Cruz-Aguado et al immobilized

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ochratoxin A on an agarose-based resin to select the aptamer to bind to the target.20

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However, this chemically modification may change their original structure and make

56

them lose some important sites for aptamers binding. To circumvent this limitation,

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some target-immobilization free SELEX protocols have been emerged, such as

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GO-SELEX21, multi-GO SELEX22, capture-SELEX23 and switching SELEX.24,25 Due

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to the adsorbtion of GO and ssDNA via π-π stacking, the unbound sequences can be

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separated from target-bound sequences. Thus, neither target nor ssDNA library was

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needed immobilized in GO-based SELEX. Both capture-SELEX and switching

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SELEX are based on ssDNA library immobilized onto magnetic beads. ssDNA library

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is

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streptavidin-coated magnetic beads. The target-bound sequences released from the

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beads due to their structure switching and therefore separated from the unbound

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sequences retained on the beads.

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designed

hybridize

to

a

complementary,

5-biotinylated

DNA on

the

CLB is the kind of small molecules with low molecular weight (313.7 g/mol) and 4


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less functional

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are appropriate for its aptamer selection. Therefore, in this work, ssDNA library

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immobilized SELEX procedure was applied to screen aptamers binding to CLB

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specifically. GO adsorption method was adopted for further research of binding

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affinity and applicability of aptamers for CLB. This ssDNA library immobilization

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SELEX protocol avoids the structure change of target and is much convenient and

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effective with the help of magnetic field. To the best of our knowledge, this is the first

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public report of aptamers selection against CLB based on ssDNA library immobilized

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SELEX. It will build a foundation for aptamer application in CLB detection and

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provide an alternative method for aptamers selection against small molecules.

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MATERIALS AND METHODS

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Materials and instruments

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groups.

Only

target-immobilization free

SELEX

protocols

The initial ssDNA random library with 80 nt was synthesized by Integrated DNA

81

Technologies (IDT) (Coralville,

IA, USA)

with the following sequence:

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5′-AGCAGCACAGAGGTCAGATG-N40-CCTATGCGTGCTACCGTGAA-3′.

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ssDNA library consists of a central 40-nt randomized sequence and 20-nt fixed primer

84

sequence at two sides. The primers used for amplification and biotin labeled strand P1

85

complemented with ssDNA library were synthesized by Sangon Biotechnology Co.,

86

Ltd. (Shanghai, China). Forward primer: 5′-AGCAGCACAGAGGTCAGATG-3′,

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phosphorylated reverse primer: 5′-(phosphate)-TTCACGGTAGCACGCATAGG-3′,

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Biotin-P1: 5′-bio-AGCACGCATAGG-3′

The

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All PCR reagents and other electrophoresis components were purchased from

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Sangon Biotechnology Co., Ltd. (Shanghai, China). Clenbuterol hydrochloride,

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ractopamine and acrylamide/bis-acrylamide 30% solution were purchased from

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Sigma–Aldrich Company (St. Louis, MO, USA). Salbutamol, epinephrine and 5



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dopamine were purchased from Aladdin Co., Ltd. (Shanghai, China). Norepinephrine

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and isoprenaline were purchased from TCI Co., Ltd. (Shanghai, China). The lambda

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exonuclease and 1×lambda exonuclease reaction buffer were purchased from New

96

England Biolabs (Hitchin, UK). Gel-red Stain solution was purchased from Botium

97

Company (California, USA).

98

Ultrapure water was from Milli Direct-Q®3 ultrapure water system (Millipore,

99

Bedford, MA). PCR amplification was conducted in a Bio-Rad C1000 Thermal

100

Cycler (Bio-Rad Co., USA). Gel electrophoresis and imaging was performed in a

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PowerPac Basic Power Supply and Molecular Imager® Gel Doc™ XR + System with

102

Image Lab™ Software (Bio-Rad Co., USA). The concentration of PCR product and

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ssDNA pool was qualified by NanoDrop 2000 Spectrophotometer (Thermo Fisher

104

Scientific, Co., USA). The fluorescence intensity of FAM-labeled ssDNA was

105

measured on an F-7000 fluorescence spectrophotometer (Hitachi Co., Japan).

106

Preparation of ssDNA library immobilized magnetic beads

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Amine-functionalized Fe3O4 magnetic beads used for ssDNA library 26

108

immobilization were prepared according to Wang and Li’s method

109

information). Then the magnetic beads coated with the avidin based on classical

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glutaraldehyde method

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immobilization, the biotin labeled complementary strand P1 (Biotin-P1) was designed

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to pair with the fixed primer area of ssDNA library. The ssDNA library was mixed

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with Biotin-P1 in the molar ratio of 1:1.5 in binding buffer (50 mM Tris-HCl, 5 mM

114

KCl, 100 mM NaCl, 1 mM MgCl2, pH 7.4). The amount of Biotin-P1 in each

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selection round was shown in Table 1. The mixture was heated at 95 °C for 10 min,

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and transferred promptly to 37 °C for 3 h to finish hybrid complementary. Then, the

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duplex with biotin label was immobilized on avidin coated magnetic beads with the

27

(see supporting

(see supporting information). For ssDNA library

6


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mass ratio of 1:80 in 37 °C for 6 h. The details were shown in Table 1. The library

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immobilized magnetic beads were washed with binding buffer for six times to remove

120

unfixed ssDNA.

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Aptamer selection

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The ssDNA library immobilized SELEX procedure applied in this study is

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illustrated in Scheme 1. In the initial selection round, 1 nmol random ssDNA library

124

were immobilized on beads and incubated with CLB (100 µM) in binding buffer at

125

37 °C for 2 h with gentle shaking. An initial incubation volume of 1 mL was used for

126

the first round, and this was decreased to 300 µL for subsequent rounds. During the

127

incubation, the sequences which bound with CLB were broken away from the duplex

128

and released from the beads. The ssDNA-CLB complexes were separated by using

129

magnetic force and collected to serve as template to be amplified by PCR. 50 µL PCR

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mixture consisted of 5 µL ssDNA template, 1 µL forward primer (5 µM), 1 µL reverse

131

primer (5 µM), 1 µL dNTP (5 mM), 0.5 µL Taq DNA polymerase (5 U/µL), 5 µL

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1×PCR buffer, 36.5 µL ultrapure water. The thermal cycle parameter was denatured at

133

94 °C for 5 min, followed by 19 cycles of denaturation at 94 °C for 30 s, annealing at

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58 °C for 30 s, and extension at 72 °C for 30 s, then extension at 72 °C for 2 min,

135

cooled at 4 °C. 8% polyacrylamide gel electrophoresis was used to separate PCR

136

products. After stained with Gelred, the gel was photographed under UV light to

137

confirm 80 bp size of PCR products. The PCR products were purified with phenol

138

chloroform method.

139

To obtain the ssDNA pool for the next selection round, phosphorylated reverse

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strand of double-stranded DNA was digested by lambda exonuclease. The

141

concentration of purified PCR product was quantified by NanoDrop 2000

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Spectrophotometer to calculate the amount of lambda exonuclease and exonuclease 7



143

reaction buffer. The digestion was conducted at 37 °C for 30 min, 75 °C for 10 min,

144

and then identified by 8% denaturing polyacrylamide gel electrophoresis. The

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digestion products were purified by phenol chloroform method and used for

146

sub-library in the next selection rounds.

147

To increase the selection pressure, experimental conditions were changed in each

148

round. As shown in Table 1, the concentration of ssDNA pool was reduced from 1000

149

pmol to 10 pmol, CLB reduced from 100 µM to 1 µM, and the incubation time

150

reduced from 120 min to 30 min from the 1st to the 16th selection rounds. Besides, to

151

eliminate nonspecific binding the counter SELEX process was employed by using

152

avidin and analogues including salbutamol, ractopamine, epinephrine, dopamine,

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norepinephrine and isoprenaline in 5th, 7th, 9th, 11th, 13th, 14th, 15th selection round.

154

These counter targets were firstly mixed with the ssDNA library immobilized beads

155

37 °C for 2 h in 300 µL binding buffer. The supernatant containing nonspecific

156

ssDNAs bound to counter targets was removed by magnetic force. Then, the beads

157

were collected and washed with binding buffer for six times and incubated with CLB.

158

The subsequent procedures were the same with the positive SELEX and the

159

experimental conditions were shown in Table 1.

160

Cloning and Sequencing

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The aptamers binding with CLB were enriched after sixteen selection rounds,

162

and the PCR products were cloned to obtain sequences by Sangon Biotechnology Co.,

163

Ltd. (Shanghai, China). The homology of sequences was analyzed with DNAMAN

164

software and their secondary structures were predicted by RNA Structure software

165

v4.60. Based on the homology and secondary structures, sequences were divided into

166

five families, and the aptamer candidates with highest enrichment and lower free

167

energy of formation △G in each family were picked out for binding assay. Then five 8


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sequences were synthesized with a carboxyfluorescein (FAM) fluorescence label at

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the 5′ end.

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Aptamer binding assay

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The affinity and specificity of five sequences binding to CLB were assessed via

172

the adsorption properties of GO. Fluorescence labeled (5′-FAM) sequences from 10

173

nM to 200 nM were incubated with CLB (1 µM) at 37 °C for 2 h in 300 µL binding

174

buffer, respectively. Then, a certain amount of GO (1.5 mg/mL) (the mass ration

175

between GO and aptamers was 70:1) was added and incubated at 37 °C for 30 min.

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The complexes of CLB and sequences were collected by centrifugation and obtained

177

for fluorescence analysis. Negative controls without CLB added were used to

178

determine nonspecific binding. The relative fluorescence intensity at different

179

concentrations of sequences was used for plotting the saturation curves and

180

calculating the dissociation constant Kd values by GraphPad Prism 5.0 software. To

181

assess the specificity, 200 nM of sequences were incubated with the avidin and

182

structural

183

norepinephrine and isoprenaline) in 300 µL binding buffer at 37 °C for 2 h

184

respectively. The concentration of each counter target was fixed at 1 µM. Then, 70 µL

185

GO (1.5 mg/mL) was added and incubated at 37 °C for 30 min to adsorb the unbound

186

aptamers. The fluorescence intensity of the complexes of CLB and sequences was

187

measured followed by centrifugation.

188

Detection of CLB using aptamer-based fluorescent analysis

analogues

(salbutamol,

ractopamine,

epinephrine,

dopamine,

189

An aptamer-based fluorescent assay was established for CLB detection to prove

190

the potential application of aptamers in real samples. 30 µL of FAM labeled aptamer

191

CLB-2 (1 µM), 30 µL of CLB with different concentrations, and 240 µL of binding

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buffer were mixed and incubated at 37 °C for 2 h. The same sample without CLB was 9



193

used as blank control. Then, 35 µL of GO (1.5 mg/mL) was added and incubated

194

37 °C for 30 min. The mixture was centrifuged at 14000 rpm for 15 min, and the

195

supernatant was collected and measured on an F-7000 fluorescence spectrophotometer.

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The detection of CLB in real samples was also studied. Pork was purchased from the

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local market and pretreated according to the literature with appropriate

198

modifications.28 500 g pork was homogenized for 10 min into a smooth paste. Next, 1

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mL of CLB standard solutions at different concentration (50 ng/mL, 100 ng/mL, 500

200

ng/mL) were individually mixed with 100 g pork paste and homogenized for 10 min.

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Then, 2 g of the above homogenized sample was mixed with 1 mL 0.02 M HCl and

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centrifuged at 14000 rpm for 10 min. The supernatant was adjusted pH to 7.4 by 2 M

203

NaOH (5 µL) and centrifuged at 14000 rpm for 10 min. The supernatant was further

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filtered with a 0.45 µm filtration membrane and collected to next assay.

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RESULTS AND DISCUSSION

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ssDNA library immobilized SELEX for evolution of CLB aptamers

207

The amount of ssDNA immobilized on the beads has great impacts on the library

208

variety that is important for the selection. Therefore, the amount of beads used for

209

immobilization was optimized by using UV–vis absorption spectroscopy. As shown in

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Fig. 1, a strong absorbance of aptamer can be seen at 260 nm before conjugation to

211

beads (black curve). After ssDNA library was immobilized on the beads, the

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supernatant was collected by magnetic separation. The absorbance of the supernatant

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liquor was weaker at 260 nm with the increase of beads. When the mass ratio between

214

beads and library reached to 80:1, the absorption of free ssDNA in supernatant tended

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to zero, indicating that the complementary ssDNA library were completely

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immobilized on the beads. Therefore the mass ratio of 80:1 between beads and library

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was used in ssDNA library immobilization. 10


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As the selection proceed, the selection pressure was gradually increased,

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including the reduction of ssDNA pool for immobilization, the decrease of the

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concentration of CLB and the contraction of incubation time. During the incubation,

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the bound ssDNA folded three dimensional conformations, resulting in the breakage

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of hydrogen bond between ssDNA and Biotin-P1. The bound ssDNA released from

223

beads while the unbound ssDNA were still immobilized on the beads via base pairing

224

with Biotin-P1. The ssDNA-CLB complexes were collected by magnetic separation

225

and served as template for PCR amplification. As shown in Fig. 2, the PCR products

226

of sixteen selection rounds were pure bands with the correct size in polyacrylamide

227

gel electrophoresis (PAGE) indicating the bound ssDNA were amplified successfully

228

in each rounds.

229

As the selection evolved, more ssDNA were released from beads and the

230

CLB-bound ssDNA were enriched, leading to the increase of the concentration of

231

template for amplification. As a result, the concentration of PCR products is higher

232

than that of the blank control under the same PCR conditions. Thus, the concentration

233

ratio (PCR products concentration of bound ssDNA/blank control) measured by

234

Image Lab software was used to investigate the efficiency of enrichment for each

235

round. As shown in Fig. 3, with increasing rounds of selection, the concentration ratio

236

increased, except for the fifth, seventh, ninth, eleventh, and thirteenth round, which is

237

count-SELEX round with decreased concentration ratio. The concentration ratio

238

reached to maximum at 16th round and started to saturate at 17th. Besides, the PCR

239

band of bound ssDNA was much brighter than that of the blank control in 16th and

240

17th selection round (Fig. S1). Therefore, the purified PCR products in 16th round

241

were cloned and sequenced.

242

Determination of binding affinity and specificity 11



243

Forty Sequences were obtained by cloned and sequenced, and five families were

244

grouped based on homology and secondary structures. One representative aptamer

245

candidate with highly enrichment and lower energy was synthesized with FAM label

246

for binding assay from each family. The binding assay was carried out according to

247

the adsorption of aptamer on GO which is induced by π-π stacking interaction

248

between the exposed nucleobases and GO. As shown in Fig. S2, in the presence of the

249

CLB, the conformation of aptamer candidates was changed and cannot be adsorbed

250

by GO. However, the conformation of unbound sequences were not changed and

251

adsorbed by GO. The higher the fluorescent intensity, the better affnitiy of aptamer

252

performed. As shown in Table 2, the Kd value of aptamer CLB-2 (76.61±12.70 nM)

253

were lower than other aptamers, indicating the better affinity to CLB. The saturation

254

curve and predicted secondary structure are shown in Fig. 4. Then, aptamer CLB-2

255

was evaluated by the specificity assay. As shown in Fig. 5, the relative fluorescence

256

intensity of CLB is much higher than that of avidin and other analogues, indicating

257

the high specificity of aptamer CLB-2.

258

An aptamer based fluorescent bioassay for CLB detection

259

To demonstrate the potential use of the aptamer CLB-2 in the quantitative

260

determination of CLB, a fluorescent bioassay was established based on GO adsorption

261

method. The increased fluorescence intensity generated by FAM labeled aptamer was

262

observed depending on the increased concentration of CLB. As shown in Fig. 6, a

263

strong linear correlation (y=101.46x+211.36, R2=0.9927) was obtained between the

264

relative fluorescence intensity and the concentration of CLB ranging from 0.10 ng/mL

265

to 50 ng/mL. The detection limit (LOD) was 0.07 ng/mL estimated by the equation

266

LOD = 3SD/slope, where SD represents the standard deviation of blank samples and

267

the slope was obtained from the calibration curve. 12


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The accuracy of the measurements was also evaluated by determining the

269

recovery of CLB in the spiked pork samples. As shown in Table 3, the recovery rate

270

was between 98.88 % and 109.40%, demonstrating that the developed aptasensor can

271

be applied to the quantitative determination of CLB in the real samples.

272

In this work, we successfully developed an ssDNA library immobilized SELEX

273

procedure by attaching ssDNA library to magnetic beads via a complementary DNA

274

to their constant region to screen the aptamer against CLB. This strategy is well suited

275

for small molecules aptamer selection because it would not change the native

276

structure of target. Among the aptamer candidates, CLB-2 was identified as the

277

optimal aptamer probe with the dissociation constant Kd of 76.61±12.70 nM by

278

GO-adsorption assessment. An aptamers-based fluorescent method was established to

279

confirm the potential application of the CLB-2 as high affinity and specificity

280

recognition receptors for CLB detection in pork samples. This work demonstrated the

281

feasibility of the aptamer of CLB for food safety control, and provided an alternative

282

select strategy for aptamer selection against small molecules especially those lack

283

enough sites for immobilization.

284

ASSOCIATED CONTENT

285

Supporting information

286

The synthesis and modification of amine-functionalized Fe3O4 magnetic beads, the

287

PCR band of 16th and 17th selection round (Fig. S1), binding assay procedure (Fig.

288

S2), and the optimization of GO concentration (Fig. S3).

289

AUTHOR INFORMATION

290

Corresponding author

291

* E-mail: [email protected], [email protected]

292

Funding 13



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This work was partially supported by BE2016306, the National Science and

294

Technology Support Program of China (2015BAD17B02), BK20140155, NSFC

295

(31401575),

296

Collaborative innovation center of food safety and quality control in Jiangsu Province.

297

Notes

298

The authors declare no competing financial interest.

299

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detection

15


and

pharmacokinetics


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of

amine-functionalized

magnetite

nanoparticles

17


and

hollow


Figure caption Scheme 1 The selection procedure of ssDNA library immobilized SELEX technique. Fig. 1 The optimization of the mass ratio between magnetic beads and ssDNA library for immobilization. Fig. 2 The polyacrylamide gel electrophoresis of PCR products from 1st round to 16th round. Fig. 3 The concentration ratio (PCR products concentration of bound ssDNA/blank control) measured by Image Lab software. Fig. 4 The secondary structure predicted by RNA Structure software v4.60 and the corresponding saturation curve of aptamer CLB-2. Fig. 5 The specificity evaluation of aptamer CLB-2 against CLB. Fig. 6 Standard curve between the relative fluorescence intensity and the concentrations of CLB. Table 1 The experimental conditions in aptamers selection against CLB Table 2 Sequence (5′-3′) and dissociation constants Kd values of aptamer candidates. Table 3 The recovery of CLB in pork samples by aptamer based fluorescent bioassay.

18


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Scheme 1

19



Fig. 1

20


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Fig. 2

21



Fig. 3

22


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Fig. 4

23



Fig. 5

24


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Fig. 6

25



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Table 1 The experimental conditions in aptamers selection against CLB Selection

ssDNA

Biotin-P1

Magnetic

CLB

Incubation time

Counter

counter targets

round

(pmol)

(pmol)

beads (µg)

(µM)

(min)

selection

(µM)

1

1000

1500

2000

100

120

No

--

2

100

150

200

60

120

No

--

3

100

150

200

60

120

No

--

4

100

150

200

60

120

No

--

5

100

150

200

60

120

Yes

60

6

80

120

160

40

90

No

--

7

80

120

160

40

90

Yes

40

8

80

120

160

40

90

No

--

9

60

90

120

20

90

Yes

20

10

60

90

120

20

90

No

--

11

40

60

80

10

60

Yes

10

12

40

60

80

10

60

No

--

13

20

30

40

5

40

Yes

5

14

20

30

40

5

40

Yes

5

15

10

15

20

1

30

Yes

1

16

10

15

20

1

30

No

--

26


Page 27 of 29


Table 2 Sequence (5′-3′) and dissociation constants Kd values of aptamer candidates No.

Sequences

Kd (nM)

CLB-1

AGCAGCACAGAGGTCAGATGATAATGTATTGTAATATTATAT TATAGAATTAATCAATTTCCTATGCGTGCTACCGTGAA

173.4±59.81

CLB-2

AGCAGCACAGAGGTCAGATGTCATCTGAAGTGAATGAAG GTAAACATTATTTCATTAACACCTATGCGTGCTACCGTGAA

76.61±12.70

CLB-3

AGCAGCACAGAGGTCAGATGATCCAAGTAGGTGTCACCTT AACAACTCTTTGAATTTATCCCTATGCGTGCTACCGTGAA

938.3±509.0

CLB-4

AGCAGCACAGAGGTCAGATGAATTTGCATAACAATATCAA CTGAGGATTACCCTCAGCATCCTATGCGTGCTACCGTGAA

571.8±156.0

CLB-5

AGCAGCACAGAGGTCAGATGTATGACAACATAGTCTTACA TTCTATGACATTCGTGATGCCCTATGCGTGCTACCGTGAA

347.4±161.1

27



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Table 3 The recovery of CLB in pork samples by aptamer based fluorescent bioassay Spiked concentration

Detected concentration

Recovery rates

(µg/kg)

mean ± SD (µg/kg)

(%)

0.50

0.504±0.023

100.80

1.00

1.094±0.098

109.40

5.00

4.944±0.178

98.88

28


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Graphic for table of contents

29


Selection and Application of ssDNA Aptamers against Clenbuterol

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