Selection, Identification, and Binding Mechanism Studies of an ssDNA

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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

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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

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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,

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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

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(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

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

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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

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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

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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

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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

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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

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

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

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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

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

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

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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

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

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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

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

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

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Funding

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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.

344

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.

351 352 353 354 355 356

19



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357 358

Figure 1

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359 360

Figure 2

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Page 22 of 26

A

B

363 364

Figure 3

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Page 23 of 26


366 367

Figure 4

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Page 24 of 26

369 370

Figure 5

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Page 25 of 26


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Figure 6

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Graphic for table of contents

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Selection, Identification, and Binding Mechanism Studies of an ssDNA

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