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Food Safety and Toxicology

Selection , Identification and Binding mechanism Studies of an ssDNA Aptamer Targeted to different stages of E.coli O157:H7 Ying Zou, Nuo Duan, Shijia Wu, Mofei Shen, and Zhouping Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01006 • Publication Date (Web): 14 May 2018 Downloaded from http://pubs.acs.org on May 15, 2018

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Journal of Agricultural and Food Chemistry

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Selection, Identification and Binding mechanism Studies of an ssDNA

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Aptamer Targeted to different stages of E. coli O157:H7

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Ying Zou, ab Nuo Duan, ab* Shijia Wu, ab Mofei Shen, ab Zhouping Wang abcd*

5 a

6

State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China

7 8

b

School of Food Science and Technology, Jiangnan University, Wuxi 214122, China

9 c

10

International Joint Laboratory on Food Safety, Jiangnan University, Wuxi 214122, China

11 12 13

d

Collaborative innovation center of food safety and quality control in Jiangsu Province, Jiangnan University, Wuxi 214122, China

14

*To whom correspondence should be addressed

15

Tel: +86 510 85917023

16

Fax: +86 510 85917023

17

E-mail: [email protected]; [email protected]

18

1

ACS Paragon Plus Environment


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ABSTRACT: Enterohemorrhagic Escherichia coli O157:H7 (E. coli O157:H7) is

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known as an important food-borne pathogens related to public health. In this study,

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aptamers which could bind to different stages of E. coli O157:H7 (adjustment phase,

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log phase and stationary phase) with high affinity and specificity was obtained by

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Whole Cell-SELEX method through fourteen selection rounds including three

24

counter-selection rounds. Altogether, thirty-two sequences were obtained and nine

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families were classified to select the optimal aptamer. To analyze affinity and

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specificity by flow cytometer, an ssDNA aptamer named Apt-5 was picked out as the

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optimal aptamer that recognize different stages of E. coli O157:H7 specifically with

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the Kd value of 9.04 ± 2.80 nM. In addition, in order to study the binding mechanism,

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target bacteria were treated by proteinase K and trypsin, indicating that the specific

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binding site is not protein on cells membrane. Furthermore, we treated E. coli

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O157:H7 with EDTA, the result showed that the binding site might be

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lipopolysaccharide (LPS) on outer membrane of E. coli O157:H7.

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KEYWORDS: aptamer, E. coli O157:H7, SELEX, flow cytometry, proteinase

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INTRODUCTION:

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Enterohemorrhagic Escherichia coli (EHEC) is an important food-borne

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pathogens related to public health, it has posed serious public health hazard all around

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the world. E. coli O157 is the most frequently studied serotype and most potent

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among EHEC.1,2 It can cause haemolytic uraemic syndrome (HUS) mainly by

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secretion of Shiga toxins encoded by the genes stx1 or stx2 and variants.3-5 Therefore,

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it is quite important to develop a rapid, sensitive and cost-effective analysis method

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for E. coli O157:H7.

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Aptamer is a short single-stranded DNA (ssDNA) or RNA sequence selected by

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systematic evolution of ligands by an exponential enrichment (SELEX), which was

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initially introduced by the groups of Ellington6 and Tuerk7. Aptamers can be selected

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for a variety of targets from small molecules to whole organisms, including ions,8,9

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toxins10-12 and pathogens13,14 related to food safety. Aptamers show the following

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significant advantages compared with antibodies: rapid and efficient recognition,

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economical and facile preparation, a wide range of targets, stability during storage and

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functionalization with flexibility. Owing to its excellent characters with high

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sensitivity and high specificity, aptamer can be used as recognition elements in many

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fields of detection system.

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Aptamers against E. coli O157:H7 have been reported in these years. Young Ju

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Lee15 used Cell-based subtractive SELEX technology obtained an RNA aptamer with

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Kd value of 110 nM through 6 rounds of selection, which RNA aptamers against E.

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coli O157:H7 was originally reported. Compared to DNA aptamers, RNA aptamers

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have more flexible structures, which allow them for more complex folding and more

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diverse configurations with target molecules, with unique advantages in the field of

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small molecule detection. However, non-chemically modified RNA aptamers were

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degraded rapidly because of their poor biological stability. Thus the production of

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RNA aptamers is more expensive and rather complex compared with ssDNA due to

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the necessity of modification for stabilization. Masoum Amraee16 also used

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subtractive SELEX technology to obtain an ssDNA aptamer against E. coli O157:H7

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through six rounds.

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In this study, we applied the Whole Cell-SELEX technique to select aptamers

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against E. coli O157:H7. Considering that bacterial outer membrane structure may be

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different at different periods, we used different periods E. coli O157:H7 (adjustment

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phase, log phase and stationary phase) as multi-target, so that in the actual sample

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detection, the aptamer can accurately identify the bacteria in every period and

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improve the accuracy in detection. Through fourteen selection rounds, an aptamer that

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can recognize different stages of E.coli O157:H7 specifically with the Kd value of

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9.04 ± 2.80 nM was obatained.

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However, we didn’t know the specific binding site between aptamers and

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bacteria due to the Whole Cell-SELEX technique. In order to study the binding

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mechanism, we did a further research on E. coli O157:H7. We treated the bacteria

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with proteinase and EDTA to see where the specific binding site might be. This is the

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first time to report DNA aptamer against different stages of E. coli O157:H7 and

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do preliminary study on binding mechanism. It provides theoretical guidance for the

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design, screening and performance evaluation of aptamers. The results have a positive

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effect on aptamers’ application.

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MATERIALS AND METHODS

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Bacterial strains and reagents. The bacterial strains used for experiment were

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obtained from the American Type Culture Collection (ATCC). E. coli O157:H7

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(ATCC 03820) was used as the target strain, Salmonella typhimurium (ATCC 10420),

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Staphylococcus aureus (ATCC 29213), Escherichia coli (ATCC 25922), Shigella

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flexneri

87

jiagang

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counter strains, and also for specificity studies. All bacteria were grown in Luria

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Bertani (LB) culture medium (10 g peptone, 5 g NaCl and 5 g yeast extract per 1 L,

90

pH 7.2-7.4) under aerobic conditions at 37℃, and all liquid cultures were shaken at

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120 rpm.

(ATCC

12022),

Escherichia coli

ETEC

(Provided

by

Zhang

Entry-Exit Inspection and Quarantine Bureau) were used as negative,

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All chemicals for preparing the buffers and solutions were purchased from

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Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Bacteria were washed

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before and after incubation with 1×binding buffer (1×BB，50 mmol/L Tris-HCl (pH

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7.4), 5 mmol/L KCl, 100 mmol/L NaCl, 1 mmol/L MgCl2). Buffer used for selection

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was prepared by adding excessive yeast tRNA (purchased from Sigma-Aldrich

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Company) and BSA (1 mg/mL) into binding buffer to reduce background binding.

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1×TE buffer (10 mmol/L Tris-HCl，1 mmol/L EDTA，pH 7.4) was used for dissolving

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ssDNA and primers. All the buffers must be sterilized in case of contamination.

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The PCR components including PCR buffer, dNTPs and Taq DNA polymerase

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were purchased from Shanghai Sangon Biological Science & Technology Company

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(Shanghai, China). The lambda exonuclease and 1×lambda exonuclease reaction

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buffer were purchased from New England Biolabs (Hitchin, UK). The polyacrylamide

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gel electrophoresis (PAGE) components, such as the acrylamide/bis-acrylamide 30%

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solution, were purchased from Sigma-Aldrich Company (St. Louis, MO, USA), and

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both ammonium persulfate and TEMED were purchased from Shanghai Sangon

107

(Shanghai, China). The solutions were prepared with ultra-high purity water from a

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Millipore water purification system.

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Initial ssDNA library and primers. The initial ssDNA library was synthesized

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by Integrated DNA Technologies (IDT). A single stranded 80-mer consists a central

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randomised sequence of 40 nucleotides (nt) flanked by two primer hybridization sites

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(5’-TGA GCC CAA GCC CTG GTA TG-N40-GGC AGG TCT ACT TTG GGA

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TC-3’). The primers used for amplification were synthesized by Shanghai Sangon

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(Shanghai, China). Based on the standard primer considerations, FAM-labeled

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forward primer and phosphorylated reverse primer were designed as (5’-FAM-TGA

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GCC CAA GCC CTG GTA TG-3’) and (5’-P-GAT CCC AAA GTA GAC CTG

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CC-3’), respectively.

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Bacterial culture. In order to obtain target bacteria in different phases, the

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growth curve of bacteria was determined. E. coli O157:H7 were grown in LB culture

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medium under aerobic conditions at 37˚C with shaking at 120 rpm. The growth rate

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was traced by measuring the optical density (OD) at 600 nm using a UV-1800

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spectrophotometer. Then drawing a growth curve of E. coli O157:H7 (Figure 1) based

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on the data. To get multi-target, different stages of bacteria (OD600 = 0.1, OD600 = 0.5,

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OD600 = 1.3) were obtained based on growth curve, then centrifugated at 5000 rpm for

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5 min to remove the supernatant and wash two times, re-suspended in binding buffer

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to a density of 108 cfu/mL in preparation for SELEX selection.

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In vitro selection. Aptamer candidates against E. coli O157:H7 were selected

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using the SELEX protocol based on Duan17,18 with slight modifications. Before

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selection the ssDNA library was denatured by heating at 95˚C for 5 min then snap

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cooled on ice for 10 min to prevent intra-strand base pairing. For the first round,

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SELEX was initiated with 1 nmol of random ssDNA library and incubated with a total

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of 108 E. coli O157:H7 cells in binding buffer at 37˚C for 1 h with gentle shaking. An

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excess of tRNA and BSA was added to the incubation buffer to reduce nonspecific

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binding. The tRNA is present to compete with the aptamer sequences for target

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binding sites. Following incubation, the cells were centrifuged at 5000 rpm at 4˚C for

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5 min, the supernatants were removed, and the cells were washed twice in 300 µL of

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1× BB before a final resuspension in 300 µL of 1× PCR reaction buffer. The cells were

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then heated at 95˚C for 10 min and placed on ice for 10 min to denature and elute

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cell-bound aptamers. The mixture was then centrifuged at 8000 rpm at 4˚C for 8 min,

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and the supernatant was isolated and designated the cell-bound aptamer fraction. All

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fractions collected were amplified by PCR.

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A total of 50 µL PCR mixture consisted of 1 µL of ssDNA template, 0.5 µL of

143

forward primer (10 µM ), 0.5 µL of reverse primer (10 µM ), 1 µL of dNTP (5 mM ), 0.5

144

µL of Taq DNA polymerase (5 U/ µL ), 5 µL of 10×PCR buffer, and 42.5 µL of

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ultrapure water. The thermal cycle parameter was denatured at 95˚C for 5 min,

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followed by 16 cycles of denaturation at 95˚C for 30 s, annealing at 56˚C for 30 s, and

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extension at 72˚C for 30 s, then extension at 72˚C for 1 min and cooled at 4˚C. Next,

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8% polyacrylamide gel electrophoresis was used to verify PCR products. After being

149

stained with Gel-red, the gel was photographed under UV light to confirm the 80 bp

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size of PCR products. The PCR products were purified with a Generay PCR

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Purification Kit.

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

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

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concentration of purified PCR product was quantified by a NanoDrop 2000

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spectrophotometer to calculate the amount of lambda exonuclease and exonuclease

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reaction buffer. The digestion was conducted at 37˚C for 40 min, 75˚C for 10 min, and

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

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products were purified by a phenol chloroform method and used for the sublibrary in

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the next selection rounds. The selection procedure was repeated until the fourteen

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round of PCR amplification including three counter selection rounds was completed.

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Cloned, sequencing and analysis of sequences. After the 14th round of

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selection, the products were cloned to obtain sequences by Sangon Biotechnology

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

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software, and their secondary structures were predicted by Mfold software. Based on

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the homology and secondary structures, thirty-two sequences were divided into nine

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families, and the aptamer candidates with highest enrichment and lower free energy of

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formation ∆G in each family were picked out for the binding assay. Then nine

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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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Identification of affinity and specificity by flow cytometer. Nine fluorescently

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labeled aptamer sequences were incubated with E. coli O157:H7 at 37˚C for 90 min

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and were analyzed by flow cytometry. To determine the binding affinity of the

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selected aptamers to the target E. coli O157:H7 cells, we performed binding assays as

174

described above with increasing amounts of a single aptamer (from 10 nM to 200 nM)

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and a constant amount of cells (108 cfu/mL) for each assay. After obtaining

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fluorescence measurements, saturation curves were calculated from these data and the

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dissociation constant Kd was calculated by nonlinear regression analysis. In order to

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identify the affinity of aptamer with each growth stage, different stages bacteria

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(OD600 = 0.1, OD600 = 0.5, OD600 = 1.3) were incubated with fluorescently labeled

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aptamer, respectively.

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Proteinase Treatment for Bacteria. To study the binding mechanism, E.coli

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O157:H7 were treated by trypsin and proteinase K .The procedure was based on a

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previously published method19,20 with some modifications,

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(OD600 = 0.1, OD600 = 0.5, OD600 = 1.3) were obtained, centrifugated at 5000 rpm for 5

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min to remove the supernatant and wash two times, then incubated with 1 mL of

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0.25% trypsin and 0.1 mg/mL proteinase K in 1×BB at 37˚C for 10 and 30 min

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respectively. After incubation, the mixture was centrifugated and washed to remove

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excess enzyme, and the treated bacteria were incubated with 100 nM FAM-labeled

different stages bacteria

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aptamer for further flow-cytometric analysis. Furthermore, in order to do a further

190

research on binding mechanism, E. coli O157:H7 were treated by EDTA according to

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Loretta Leive21 method with slight improvement. Different stages bacteria (OD600 =

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0.1, OD600 = 0.5, OD600 = 1.3) were obtained, centrifugated at 5000 rpm for 5 min,

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then incubated with 5 mM EDTA for 5 min at 37˚C, the incubation was terminated by

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adding MgCl2. After centrifugating and washing, incubated with 100 nM

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FAM-labeled aptamer for further flow-cytometric analysis finally.

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RESULTS AND DISCUSSION

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Aptamer Selection. In order to improve the affinity and specificity of the

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aptamer selection, the experimental conditions were varied with the increase of

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selection round, ssDNA pool was reduced from 1 nmol to 100 pmol, the amount of

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tRNA and BSA increased from a 20-fold molar excess of each in the initial round to a

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maximum of 80-fold molar excess in round eight. Increased amounts of BSA/tRNA

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increased the competition between the desired targets (cells) and non-targets (BSA

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molecules) for the aptamer molecules, tRNA competes with the aptamer sequences for

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target binding sites, leading to higher specificity. Besides, counter selection was done

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in 5, 9 and 12 rounds with a mixture of related intact pathogenic bacteria (Salmonella

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typhimurium,

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Escherichia coli ETEC). The products of each round for selection was monitored

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using flow cytometry. As shown in Figure 2, with increasing rounds of selection, The

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fluorescence intensity gradually increased, except for the counter-SELEX round. The

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decreased fluorescence intensity may be resulted from a part of ssDNA bound with

Staphylococcus

aureus,

Escherichia

coli, Shigella

flexneri,

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counter-targets in counter-SELEX. The selection was finished in the fourteenth round

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when we observed that the fluorescence intensity started to saturate and reached a

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maximum average of 76.70% at fourteenth round. Therefore, the PCR products from

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the fourteenth round were cloned and sequenced.

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Binding affinity and specificity. Altogether, thirty-two sequences were obtained

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by cloning and sequencing, and according to homology and secondary structures, nine

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families were classified. One representative aptamer candidate with high enrichment

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and lower energy was synthesized with a FAM label for the binding assay from each

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family. From the Kd values results shown in Table 1, it is demonstrated that, among

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nine candidate aptamers, five of them (Apt-1、Apt-3、Apt-4、Apt-5、Apt-7) show the

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stronger binding affinity with E. coli O157:H7. Therefore, these five aptamers were

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selected as the aptamer probe against E. coli O157:H7 for the following specificity

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detections. To characterise the specificity of the selected aptamer, FAM-labelled five

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aptamers were tested against a variety of other bacteria(108 cfu/mL,OD600 = 0.3),

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including Salmonella typhimurium , Staphylococcus aureus , Escherichia coli ,

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Shigella flexneri , and Escherichia coli ETEC. As shown in Figure 3, both sequence

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Apt-4 and Apt-5 showed preferential binding to E. coli O157:H7 with the

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average flourescence intensity approaching 80%, but sequence Apt-4 appeared

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higher binding to other bacteria, especially Salmonella typhimurium and Escherichia

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coli. By contrast, the binding affinity of sequence Apt-5 to other counter-targets was

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all below 16%. Thus, aptamer Apt-5 was determined as the optimal aptamer for E.

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coli O157:H7 due to the lowest Kd values and better specificity. The saturation curve

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and predicted secondary structure of aptamer Apt-5 are shown in Figure 4.

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In order to identify the affinity of aptamer with each growth stage, different

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stages bacteria (OD600 = 0.1, OD600 = 0.5, OD600 = 1.3) were incubated with

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fluorescently labeled aptamer, respectively. As shown in Figure 5, the result shows

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that Apt-5 has good affinity to all different stages bacteria (adjustment phase, log

238

phase and stationary phase).

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The binding mechanism. We obtained the high affinity and specificity aptamer

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Apt-5 by Whole Cell-SELEX technique. However, we didn’t know the specific

241

binding site between Apt-5 and bacteria. In order to study the binding mechanism, we

242

did a further research on E. coli O157:H7 to explore the binding mechanism. We

243

treated the bacteria with proteinase K and trypsin to destroy bacterial surface

244

membrane protein. If the fluorescence signal reduces, the target of the aptamer may be

245

protein molecules on bacterial cell wall. However, as Figure 6A shows, when treated

246

by proteinase K and trypsin (15 min) , the signal did not change too much than

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untreated, and with the increasing of treatment duration (30 min), the signal still keep

248

the same fluorescence intensity than untreated, this result suggested that the target

249

might not be membrane protein, but other cell wall composition. In order to

250

continue to study the binding mechanism, we treated E. coli O157:H7 with EDTA.

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According to Loretta Leive21 study, EDTA treatment makes LPS released, leading to

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the remain LPS on membrane reduced. As the Figure 6B shown, the signal is weaker

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than untreated, thus we deduce that the target might be LPS.

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In conclusion，this study described using whole-bacterium SELEX to screen the

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DNA aptamer with high affinity and specificity for different stages of E. coli

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O157:H7, it is the first report of using different stages of E. coli O157:H7 as targets

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and doing a further research on binding mechanism. Considering that bacteria in

258

different phases may have different surface membrane properties, we used different

259

stages of E. coli O157:H7 as target each round so that the aptamer can bind to any

260

times of E. coli O157:H7, expanding the range of targets and improving the accuracy

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in the actual sample detection. In addition, we did a further research on binding

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mechanism, the experiments with proteinase and EDTA treatment suggest that the

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target of the aptamer might not be membrane protein molecules but LPS. The work

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described in this study demonstrates the ability of whole-bacteria SELEX to screen an

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excellent aptamer probe to detect the existence of E. coli O157:H7 and has the

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potential in food safety control.

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AUTHOR INFORMATION

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Corresponding Author

270

*Tel: +86 510 85917023, Fax: +86 510 85917023

271

E-mail: [email protected]; [email protected]

272

Funding

273

This work was partially supported by Key Research and Development Program of

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Jiangsu Province (BE2016306), National Nature Science Foundation of China (NSFC

275

31401575), and Project funded by China Postdoctoral Science Foundation

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(2017M610299, 2016T90430).

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Figure legends:

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Figure 1. Growth curve of E. coli O157: H7.

338

Figure 2. The fluorescence intensity of each seletion round.

339

Figure 3. Identification of the specificity of Apt-5 against E. coli O157:H7: (A)

340

histogram of the percent gated fluorescence intensity above library background for

341

individual aptamers; (B) flow cytometry assay for the binding of aptamers to bacteria.

342

Figure 4. The secondary structure predicted by Mfold software and corresponding

343

saturation curve of aptamer Apt-5.

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Figure 5. Identification of the affinity of Apt-5 against three different stages E. coli

345

O157:H7.

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Figure 6. Binding mechanism studies of Apt-5 against three different stages E. coli

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O157:H7: (A) Treatment with proteinase K and trypsin for 15 min and 30 min,

348

respectively; (B) Treatment with EDTA.

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Table 1. Sequence (5’-3’) and dissociation constants Kd values of aptamer candidates.

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Figure 1

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359 360

Figure 2

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A

B

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Figure 3

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366 367

Figure 4

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369 370

Figure 5

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Figure 6

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Graphic for table of contents

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Selection, Identification, and Binding Mechanism ... - ACS Publications

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