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Food Sensing: Selection and Characterization of DNA Aptamers to Alicyclobacillus Spores for Trapping and Detection from Orange Juice Tim Hünniger, Christin Fischer, Hauke Wessels, Antonia Hoffmann, Angelika Paschke-Kratzin, Ilka Haase, and Markus Fischer J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 31 Jan 2015 Downloaded from http://pubs.acs.org on February 5, 2015

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Journal of Agricultural and Food Chemistry

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Food Sensing: Selection and Characterization of DNA Aptamers to

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Alicyclobacillus Spores for Trapping and Detection from Orange Juice

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Tim Hünniger, Christin Fischer, Hauke Wessels, Antonia Hoffmann, Angelika Paschke-Kratzin,

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Ilka Haase, and Markus Fischer*

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HAMBURG SCHOOL OF FOOD SCIENCE; Institute of Food Chemistry, University of Hamburg,

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Grindelallee 117, 20146 Hamburg, Germany, *Corresponding author: Tel.: +49-40-

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428384357; Fax: +49-40-428384342; E-Mail: [email protected]

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ABSTRACT

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The quality of the beverage industry’s products has to be constantly monitored to fulfil

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consumer’s high expectations. The thermo-acidophilic Gram positive Alicyclobacillus spp. are

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not pathogenic but due to their heat-resistant endospores they can survive the juice

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processing conditions and have become a major economic concern to the fruit juice

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industry. Current detection methods rely on cultivation, isolation, and organism

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identification which can take up to a week, resulting in economic loss.

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This work presents the selection and identification of DNA aptamers targeting

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Alicyclobacillus spores by spore-SELEX (systematic evolution of ligands by exponential

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enrichment) in orange juice simulating buffer. The selection process was verified by various

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techniques including flow cytometric binding assays, radioactive binding assays and agarose

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gel electrophoresis (AGE). The subsequent aptamer characterization includes the

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determination of dissociations constants (KD values) and selectivity by different techniques

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e.g. SPR and fluorescence microscopy. In summary, 10 different aptamers with an affinity to

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Alicyclobacillus spp. have been developed, analyzed and characterized towards affinity and

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specificity.

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KEYWORDS

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SELEX, spores, aptamers, Alicyclobacillus, selection, SPR, FACS, enrichment, juice

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INTRODUCTION

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Alicyclobacillus spp. are ubiquitous, non-pathogen, Gram positive, rod-shaped and

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endospore-forming microorganisms which grow at temperatures of 25-60 °C under highly

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acidic conditions (pH: 2.5 - 6.0).1-3 Some Alicyclobacillus spp., especially Alicyclobacillus

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acidoterrestris are becoming more and more a frequent challenge for fruit beverage industry

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because the metabolic products 2-methoxyphenol (guaiacol), 2,6-dibromophenol and 2,6-

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dichlorophenol induce even in small amounts an antiseptical or medicinal off-flavor.4, 5 While

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vegetative cells could be eliminated by pasteurization, the spores persist and survive low pH

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and water activity, as well as high concentrations of sugar and organic acids.6-8 The state-of-

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the-art technologies to detect Alicyclobacillus spp.-contaminations after pasteurization are

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microbiological enrichments by cultivation which are described by the International

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Federation of Fruit Juice Producers (IFU), the Australian Fruit Juice Association (AJA) and the

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Japan Fruit Juice Association (JFJA).3, 5, 9 Because of low division rates of Alicyclobacillus spp.

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this procedure can take up to a week and therefore lead to economic losses.1

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The high affinity and specificity of antibodies could be used in alternatives to microbiological

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plating methods by coupling them to solid-phases (immunoaffinity chromatography) or

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surfaces of magnetic nanoparticles (immunomagnetic nanoparticles, IMPs).1,

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other reasons, the use of animals during antibody manufacturing leads to relatively high

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production costs, volatile reproducibility and physiological restrictions. Furthermore,

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directive 2010/63/EU limits the usage of animals for scientific purposes.12

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To overcome the mentioned problems, the utilization of single stranded nucleic acids, so

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called aptamers, could be an alternative.13,

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strand, these oligonucleotides form distinct three-dimensional structures and interact with

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various targets in a highly specific way.15,

14

16

10, 11

Among

Due to the absence of the complementary

The binding affinities and specificities of 3

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antibodies and aptamers are comparable but considering the wider target range (ions,

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molecules, and cells; toxic and endogenous substances), higher physical and chemical

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stability, lower production costs, and broader availability of targeted modifications, these

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artificial acceptors expand the application area of biosensors significantly.10, 17-19

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For the development of aptamers an in vitro process called systematic evolution of ligands

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by exponential enrichment (SELEX) has to be performed (Figure 1).20 The procedure starts

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with an aptamer pool containing a randomized sequence and sidewise primer binding sites.

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The process is characterized by the steps of (i) incubation of aptamer pool and target, (ii)

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several washing steps, (iii) amplification of reduced aptamer pool (subpool), and (iv) strand

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separation to isolate single stranded aptamers. Aptamer sequences with desired binding

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properties result from 10-20 consecutive repeats of the mentioned procedure, in which the

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obtained single stranded sub pools serve as the pool for the following round.12

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To perform the substeps incubation (i) and washing (ii) during the SELEX process, various

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techniques with different advantages have been developed. A common application is the

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coupling of the target to magnetic beads, an implementation which offers excellent

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possibilities for automatization.12,

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electrophoresis-SELEX), FACS-SELEX (fluorescence-activated cell sorting-SELEX), or Just-in-

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Time-Selection (semiautomated two-step SELEX) have been developed, but the field is a part

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of current research and further development could be expected.12, 23, 24

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Although aptamer amplification is established, common problems are (i) preference of short

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DNA fragments during amplification and (ii) highly diverse aptamer pools which lead to

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undefined products by recombination of homologous template regions and formation of

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heteroduplexes.25,

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results, which is achieved by generating several different reaction compartments and ideal

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21,

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Other techniques like CE-SELEX (capillary

Utilization of emulsion PCR (emPCR) provides significantly improved


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statically distribution of reaction compounds and templates.27 To isolate single-stranded

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aptamers, the double-stranded nucleic acids have to be separated by utilization of a

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biotinylated reverse primer and streptavidin-coated magnetic beads.

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In the present study aptamers with an affinity to Alicyclobacillus spp. spores were selected in

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orange juice simulating buffer. The resulting aptamers will be used for selective trapping and

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enrichment of spores from orange juice to supersede the classical time consuming

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cultivation process. Hence, the aptamer based trapping technique (e.g. magnetic separation)

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will help to accelerate the detection of these spores. The SELEX was verified by various

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techniques including fluorescence-activated cell sorting (FACS), radioactive binding assays

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and agarose gel electrophoresis (AGE). Aptamer characterization including determination of

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dissociations constants (KD values) and selectivity were performed by surface plasmon

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resonance (SPR) spectroscopy as well as fluorometric binding assay, flow cytometric binding

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assay and fluorescence microscopy to validate obtained results.

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MATERIALS AND METHODS

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Aptamer Selection. Bacterial Strains. For the spore-SELEX Alicyclobacillus acidoterrestris

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spores (DSM 2498: 108 cfu/mL and a wild type: 108 cfu/mL) were received like all other

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spores by Lehrstuhl für Hygiene und Technologie der Milch, Tierärtzliche Fakultät, Ludwig-

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Maximilians-Universität (Munich, Germany) in paraformaldehyde (2 %) and used as

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following as SELEX target: 1/1, v/v, final concentration 107 cfu/mL. The vegetative

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Alicyclobacillus cells used for counter selection during the first four SELEX rounds comprised

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hundred undefined wild Alicyclobacillus strains and were obtained by Dr. B. Schütze, LADR

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GmbH (Geesthacht, Germany).



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Aptamer pool. The used aptamer pool for selection round one consist of following

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sequences: 5´-CATCCGTCACACCTGCTC-(N)40-GGTGTTGGCTCCCGTATC-3´ (Life technologies

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GmbH, Darmstadt, Germany).

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Aptamer Amplification and AGE. The emPCR was performed using the commercially

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available Micellula short DNA Emulsion & Purification kit (Roboklon GmbH, Berlin, Germany).

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The amplifications were carried out using biotinylated reverse primer for subsequent strand

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separation with following sequences: 5´-CATCCGTCACACCTGCTC-3´ (forward primer, Life

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technologies GmbH, Darmstadt, Germany) and 5´-Biotin-GATACGGGAGCCAACACC-3´

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(reverse primer, Life technologies GmbH, Darmstadt, Germany). Aptamer amplification was

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carried out with a volume of 100 µL of aqueous phase in a 2 mL reaction tube as follows:

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5 µL DreamTaq-Polymerase (0.5 U/µL, Fisher Scientific-Germany GmbH, Schwerte,

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Germany), 10 µL 10x DreamTaq buffer (Fisher Scientific-Germany GmbH, Schwerte,

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Germany), 8 µL dNTPs (10 mM, Bioline GmbH, Luckenwalde, Germany), 4 μL of each primer

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(100 µM, Life Technologies GmbH, Carlsbad, USA) and template varied between 20 and

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50 ng. After assembling the aqueous phase, the oil phase compounds were added

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subsequently to the reaction tube according to the manufacturer’s protocol. The tube was

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mixed 5 min at room temperature to create a homogenous emulsion. Due to the volume of

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the reaction mixture, splitting into aliquots was necessary to perform a PCR under the

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following conditions: an initial denaturation step at 95 °C for 5 min and subsequently

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25 cycles of denaturation at 95 °C for 30 s, primer-annealing at 56 °C for 30 s, and elongation

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at 72 °C for 30 s. Thereafter, a final elongation step at 72 °C for 5 min and finally a storage

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temperature of 4 °C followed. Afterwards the emulsion was broken according to the

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manufacturer protocol, the oil phase was discarded and the obtained emPCR products were


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purified using silica columns according to the protocol provided by the manufacturer. The

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emPCR products were analyzed by AGE (agarose gel electrophoresis).

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Strand Separation. After the aptamer amplification a strand separation step is mandatory to

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obtain again single stranded DNA aptamers (subpool) for further selection rounds. Magnetic

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beads (Dynabeads M-280 Streptavidin, Invitrogen Life Technologies GmbH, Carlsbad, USA)

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were incubated for 15 min under agitation with biotinylated emPCR products and the same

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volume of 2x binding- and washing buffer (1 mM EDTA, 2 M NaCl, 10 mM Tris-HCl, pH 7.5).

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The supernatant was removed using a magnetic rack, and the beads were resuspended in

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80 μL of ddH2O before the tube was incubated 10 min at 95 °C for heat denaturation of

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double-stranded emPCR products. The heated supernatant was immediately centrifuged for

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10 s at 10500 x g and the tube was placed in a magnetic rack to agglomerate the magnetic

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beads at the bottom. The still hot supernatant contained the desired aptamers ready to be

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transferred into a new reaction tube.

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General SELEX Protocol. To obtain aptamers with an affinity to Alicyclobacillus spores the

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following SELEX protocol was used: 1 nmol of the corresponding aptamer pool (100 µM) was

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heated (5 min, 95 °C) in an orange juice simulating buffer (2 µM CaCl2, 50 mM KCl, 2 µM

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MgCl2, 40 mM Tris, pH 3.5) followed by immediately cooling (4 °C) to refold the aptamers

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and eliminate heteroduplexes. Afterwards 20 µL of mentioned vegetative cells of

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Alicyclobacillus were added for counter selection. The mixture was incubated 60 min at

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room temperature followed by centrifugation (12 min at 10500 x g). The supernatant was

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refolded as described above and incubated 60 min at room temperature with 20 µL of the

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SELEX target. Subsequently the reaction mixture was centrifuged (12 min at 10500 x g) and

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the supernatant was removed. The resulting spore pellet was washed various times with

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200 µL of 1x binding- and washing buffer, centrifuged (12 min at 10500 x g) and was 7 ACS Paragon Plus Environment


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resuspended in 35 µL ddH2O (Table 1). The obtained aptamer pool was heat eluted (5 min at

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96 °C) and the hot supernatant was transferred in a new reaction tube. Subsequently the

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obtained aptamers were amplified by emPCR followed by strand separation. For further

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SELEX rounds 40 µL of each strand separated aptamer pools (corresponding subpools) were

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used. A continuously rising stringency during the SELEX process was achieved by constantly

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increased washing steps, decreasing target concentration and incubation times (Table 1).

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After 17 selection rounds the final aptamer subpool was used for cloning and sequencing.

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Aptamer Cloning. The final aptamer subpool was ligated using a TOPO TA Cloning Kit (Life

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Technologies GmbH, Darmstadt, Germany) and cloned into competent Escherichia coli cells

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XL1. Plasmid DNA was obtained and purified using the QIAprep Spin Miniprep Kit (Qiagen

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GmbH, Hilden, Germany). Ten aptamer candidates were sequenced by using M13 universal

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primer (GATC-Biotech AG, Konstanz, Germany) (Table 2).

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Determination of Aptamer Dissociation Constants (KD) via SPR. Spore lysis. Before

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immobilization of the target, the spores were fragmented using a TissueLyser (Qiagen

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GmbH, Hilden, Germany). 40 µL of the mentioned Alicyclobacillus spores (107 cfu/mL) and

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250 mg glass beads (0.5 mm diameter, Sigma-Aldrich-Chemie, Munich, Germany) were

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diluted with 360 µL sodium acetate buffer (pH 5.0, Sierra Sensors GmbH, Hamburg,

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Germany) and the mixture was lysed twice (5 min, 30 Hz) at room temperature.

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Sensor Chip Preparation. The resulting supernatant includes the fragmented spores

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(106 cfu/mL) and was directly used for following immobilization and measurements using the

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SPR2 (Sierra Sensors GmbH, Hamburg, Germany). The HPA Sensor Chips (SPR-AS-HP, Sierra

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Sensors GmbH, Hamburg, Germany) are characterized by aliphatic hydrocarbon chains on

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the surface and therefore high hydrophobic interactions occur.28,

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were carried out at 25 °C with a flow rate of 25 µL/min using degassed orange juice

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All SPR experiments


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simulating buffer containing 0.01 % Tween 20. The HPA sensor chip was activated for 4 min

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by injecting a mixture (1/1, v/v) of isopropyl alcohol and sodium hydroxide (50 mM) on both

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channels. The chip was washed for 4 min by injecting the orange juice simulating buffer and

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subsequent the fragmented spores were injected 8 min on channel 2. As reference and to

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inhibit non-specific interactions of injected aptamers with the chip surface, BSA (50 µg/mL,

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bovine serum albumin, New England Biolabs GmbH, Frankfurt am Main, Germany) was

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immobilized by an injection for 8 min on both channels.

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SPR Measurements. The dissociation constant (KD) determinations were performed in orange

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juice simulating buffer as running buffer by injecting four different aptamer concentrations

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(2.5, 5.0, 7.5, and 10.0 µM) for 6 min of pure aptamers (Life technologies GmbH, Darmstadt,

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Germany). The obtained data were evaluated using AnalyserR2 (SierraSensors GmbH,

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Hamburg, Germany) and Scrubber (BioLogic Software Pty Ltd., Campbell, Australia) after

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subtraction of the reference signal from target signal. The sensor chip was regenerated by

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injecting regeneration buffer (100 mM hydrochloric acid) for 30 s after each aptamer

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injection. In addition the orange juice simulating buffer was injected and used besides the

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BSA reference for a double referenced calculation of dissociation constants.

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Fluorescence Microscopy. To visualize the interactions between the spores and the

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aptamers fluorescence microscopy was utilized. The visualization was carried out using the

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fluorescence correlation spectroscope Cor2™ (Carl Zeiss AG, Oberkochen, Germany). Prior to

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the measurements the aptamers were fluorescently labeled with Alexa488 by emPCR and

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strand

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CATCCGTCACACCTGCTC-3´, Life technologies GmbH, Darmstadt, Germany) and biotinylated

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reverse primer. Subsequently the aptamer concentration was determined using Nanodrop

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ND-1000 spectrometer (Thermo Fisher Scientific Oy, Vantaa, Finland) and set to 5 ng/µL.

separation

using

fluorescently

labeled

forward

primer

(5´-Alexa488-



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30 µL of a labeled mix of all obtained aptamers were added to 190 µL orange juice

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simulating buffer and subsequently incubated with 30 µL Alicyclobacillus spores (107 cfu/mL)

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for 1 h at room temperature under light exclusion. The mixture was centrifuged (12 min at

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10500 x g) and the supernatant was discarded to remove excess aptamers. The resulting

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spore pellet was washed once with 100 µL 1x binding- and washing buffer, centrifuged

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(12 min at 10500 x g) anew, and the pellet was solved in 100 µL ddH2O. The analysis was

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carried out under the following conditions: argon laser (5 mW) with 25 % output and 3 %

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transmission, 40 x lens, and wavelength 488 nm which corresponds to the absorption

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maximum of the used fluorophore Alexa488.

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Determination of Aptamer Selectivity via SPR. The aptamer selectivity was investigated

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using SPR2 by measuring the affinity of the obtained aptamers to different Bacillus spores as

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well as Clostridium spores (Table 3). For the following measurements high capacity amine

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chips (SPR-AS-HCA, Sierra Sensors GmbH, Hamburg, Germany) were used.

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Sensor Chip Preparation. The sensor chips were activated for 4 min by injecting a mixture

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(1/1, v/v) of NHS (100 mM, N-Hydroxysuccinimide, Sigma-Aldrich-Chemie, Munich,

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Germany) and EDC (400 mM, 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, Applichem

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GmbH, Darmstadt, Germany) in PBS buffer. Afterwards a streptavidin solution (50 µg/mL,

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diluted in sodium acetate buffer, pH 5.0, Sierra Sensors GmbH, Hamburg, Germany) was

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immobilized by injecting for 6 min on both channels. Subsequently the sensor surface was

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blocked for 8 min by injecting ethanolamine (1 M, Sierra Sensors GmbH, Hamburg, Germany)

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on both channels. For the determination of selectivity the aptamers were modified (5’-

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Biotin-TEG, Integrated DNA Technologies BVBA, Leuven, Belgium) and immobilized by

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injecting for approximately 8 min until a response signal of 200 RU (response unit) was

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SPR Measurements. The aptamer selectivity was investigated in orange juice simulating

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buffer containing 0.01 % Tween 20 as running buffer by injecting the spores listed in Table 3.

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The various spores were diluted to a concentration of 106 cfu/mL, fragmented as described

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above and injected for 2 min. The sensor chip was regenerated by injecting regeneration

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buffer 2 (25 mM EDTA. 1 % Tween 20, pH 12.0) for 30 s after each spore injection. In

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addition the orange juice simulating buffer was injected for a referenced calculation of

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aptamer selectivity. The obtained data were evaluated as mentioned above.

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RESULTS AND DISCUSSION

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The occurrence of Alicyclobacillus spp. spores in juice is a major concern for fruit beverage

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industries due to the formation of an antiseptical and medicinal off-flavor by these

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microorganisms even after pasteurization. The endospores germinate under given condition

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during the fruit juice production, especially in course of the dilution of juice concentrates.3

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Prior to detection by various techniques spores have to be enriched typically by cultivation.

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Alternatives to the very time consuming microbiological cultivation could be antibody-based

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or aptamer-based trapping and enrichment techniques. A prerequisite for aptamer

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applications is their selection using the so called SELEX process.

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Aptamer Selection. The specification of selection conditions, like buffer compositions and

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an increasing stringency during the selection process are required to obtain aptamers with

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affinity to the target, i.e. the spores. The separation between aptamers with high affinity to

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spores respectively vegetative cells and aptamers with little or no affinity to spores

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respectively vegetative cells was achieved by centrifugation which is common during cell-

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and spore-SELEX.30, 31 These particles accumulate by centrifugation due to higher density of

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spores and vegetative cells and entrain the aptamers with an affinity for the corresponding

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target. In this work defined Alicyclobacillus acidoterrestris spores (DSM 2498, wild type, 1/1, 11 ACS Paragon Plus Environment


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v/v) were used as selection targets because these two strains seem to be representative for

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off-flavour production in fruit juices and are frequently abundant. To simulate the crucial

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conditions which influence aptamer secondary structures (ion composition: 2 µM Ca2+,

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50 mM K+, 2 µM Mg2+ and pH: 3.5), the used buffer compounds are in terms of

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concentrations as close as possible to orange juice. Especially the pH and the ionic bindings

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between cations and the DNA phosphate backbone have a huge impact on the secondary

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structure of aptamers and therefore on the binding properties.32

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In addition a continuously rising stringency during the SELEX process was achieved by

252

constantly increasing washing steps, decreasing target concentration and incubation times

253

(Table 1). Furthermore a vegetative cell mix of hundred undefined Alicyclcobacillus spp.

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strains was used for a counter selection during the selection rounds 1 to 4. The purpose of

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this approach was to generate aptamers selectively to the spores of Alicyclobacillus spp. and

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little or no affinity to the vegetative cells. This strategy provides the opportunity to

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antagonize the enrichment of vegetative cells and therefore to inhibit false-positive results

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of the developed system.

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The progress of aptamer selection was evaluated by various techniques to ensure the rising

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affinity towards the target during the SELEX process. After each SELEX round the presence of

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aptamers was checked via agarose gel electrophoresis and furthermore the increasing

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aptamer affinity was investigated via flow cytometric and radioactive binding assays

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(Supporting Information). After 17 SELEX rounds the final aptamer subpool was ligated into

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plasmids, transferred into E.coli cells for isolation and finally sequenced.

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The ten obtained aptamers with an affinity to Alicyclobacillus spores are listed in Table 2.

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Secondary structures are predicted using mfold V4.6 and shown of AliApt1, AliApt3, AliApt5,

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and AliApt9 in Figure 2.33 12 ACS Paragon Plus Environment

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Aptamer Sequence Analysis. The secondary structure prediction was performed using

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mfold V4.6.33 Taking into account the energy-minimization method, the secondary structure

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is calculated from a given primary structure, in which different characteristics of three-

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dimensional structures, such as hairpin structures, loops or bubbles, are significant.34 In

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addition folding temperature and ion concentrations are considered for calculation of the

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most energetically favorable secondary structure.35 By comparison of the exemplary

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secondary structures (Figure 2) it could be noticed that homologous areas within similar

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structure features are observable. Furthermore some characteristic structural features were

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unique in single secondary structures. The obtained aptamer sequences (AliApt1-AliApt10)

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contain increased cytosine repeats which are possibly form stable three-dimensional

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structures, called i-motifs.36,

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quadruplexes and could be further investigated by NMR analysis. The high diversity of

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structure features led to the conclusion that the obtained aptamers are presumably binding

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to various spore surface areas and not one defined spore coat. Further secondary aptamer

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structures are shown in Supporting Information (Figure S3).

37

These structure elements are very similar to guanosine

283

Determination of Aptamer Dissociation Constants (KD) via SPR. To determine

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dissociation constants of the obtained aptamers to spores, several methods have been

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considered. For example, utilization of a flow system (QCM, quartz crystal microbalance) or

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immobilization-free techniques like MST (microscale thermophoresis) and ITC (isothermal

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titration calorimetry) have been previously used with cell lysates by Seidel et al. or Zhou et

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al. but suitable approaches for spores are not directly available.38,

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established SPR technique was adapted to determine binding parameters of the obtained

290

aptamers to the Aliyclobacillus spores.

39

Therefore, the well-



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In this context we immobilized aptamers with different modifications on various sensor chip

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surfaces and injected spore suspensions without receiving evaluable data. It seems to be

293

that the considerable differences in weights of the interacting partners and utilization of a

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flow system are responsible for steric hindrances and therefore non-evaluable data. Hence,

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like in the MST- and ITC-based techniques mentioned above, various methods using

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immobilized spores and spore lysates were investigated whereby the utilization of a

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TissueLyser and glass beads enabled the preparation of an appropriate SPR sensor chip. In

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this context the optimized parameters mentioned above (glass beads with several

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diameters, sodium acetate with different pH values, and the treatment time obviously) led

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to a suitable spores fragmentation degree for a subsequent immobilization on a sensor chip.

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In addition several sensor chip surfaces (e.g. carboxylated dextran, carboxylated, aliphatic

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hydrocarbon chain) were examined whereas the aliphatic hydrocarbon chains led to the

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most appropriate SPR sensorgrams with response signals of approximately 300-700 RU

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during immobilization. According to Karlsson one response unit corresponds roughly to

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1 pg/mm2 immobilized ligand on SPR sensor chips.40 Considering the utilized sensor chip

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surface of 1.2 mm2, a total weight of 360 - 840 pg fragmented spores has been immobilized.

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Due to the high amount of immobilized ligands it can be assumed that also spore coat

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fragments have been immobilized, therefore the developed SPR immobilization method of

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spore lysate was applicable for determination of aptamer dissociation constants to

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Alicyclobacillus spores. The availability of spore coat fragments is a crucial prerequisite for

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SPR measurements due to the usage of whole spores during the SELEX process. It seems

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reasonable that the obtained aptamers possess an affinity to several coat compounds.

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Furthermore, the developed surface regeneration procedure including short contact times

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and minor chemical loads is capable to perform consecutive measurements without losses in 14 ACS Paragon Plus Environment

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response. Chip activity and stability were investigated by repeated injection of an aptamer

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(10 µM) and 50 subsequent regenerations (data not shown). The chip surface stability was

317

decreased of approximately 10 % which is less than the recommended cut-off value of

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20 %.41, 42 In summary, the implemented spore immobilization procedure and regeneration

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fulfill the requirements of appropriate SPR measurements.

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The determination of aptamer dissociations constants were carried out using orange juice

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simulating buffer to retain the secondary structures of aptamers and therefore the binding

322

properties to the Alicyclobacillus spores under conditions which are comparable to orange

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juices. Here, the aptamers were injected on multiple SPR sensor chips for 6 min in rising

324

concentrations (2.5, 5.0, 7.5, and 10.0 µM). To obtain double referenced data, the RU signals

325

of reference (BSA) and orange juice simulating buffer were substracted. Examples of

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obtained sensorgram overlays of four different aptamer concentrations (2.5, 5, 7.5, and

327

10 µM) including the corresponding fits are shown in Figure 3. For calculation of dissociation

328

constants the obtained sensorgrams were fitted with Langmuir 1:1 model including a mass

329

transport limitation factor and separately calculated for each aptamer. Dixon’s outliers were

330

eliminated and the averages of different SPR measurements were formed (Table 2). The

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obtained KD values are in the low-nanomolar range (from 2.3 to 149.1 nM) which is quite

332

comparable to published aptamer for cells and proteins.12, 19, 43

333

Due to the mentioned SPR surface chip preparation using hydrophobic adsorption of spore

334

lysates side effects like e.g. heterogeneous ligand sites, non-specific binding and charge

335

related attraction will influence binding kinetics of the injected aptamers. In contrast to a 1:1

336

interaction the obtained SPR sensorgrams for each aptamer injection reflect more a

337

multivalent binding kinetic which explains the deviation of raw data and kinetic fits using a

338

Langmuir 1:1 model including a mass transport limitation factor (Figure 3). 15 ACS Paragon Plus Environment


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339

A binding model including all side effects of aptamer spore interactions on a heterogeneous

340

surface, like a spore lysate, is currently not available but usage of a 1:1 model including a

341

mass transport limitation factor seems to be a sufficient approach of the kinetic constants.

342

However, the obtained data are indicating a specific and concentration-dependent

343

interaction of generated aptamers to the Alicyclobacillus spores, but should be regarded

344

with reservations based on the mentioned aspects. In addition, a fluorometric binding assay

345

was performed by using fluorescently labeled aptamers to confirm the assumed KD values.

346

Dissociation constants in a low-nanomolar range resulted from the fluorometric assay which

347

supports the SPR measurements (data not shown).

348

Determination of Aptamer Selectivity via SPR. The determination of dissociation

349

constants revealed four promising candidates with KD-values in the low-nanomolar range

350

(AliApt1, AliApt3, AliApt5, and AliApt9; Table 2). Selectivity is a crucial parameter for

351

aptamer evaluation, as not only Alicyclobacillus spp. but also Bacillus spp. and Clostridium

352

spp. occur as contaminants in fruit juice industry. Due to the high number of various

353

endospores (Table 3) and the high price of SPR consumables it was advantageous to develop

354

a suitable aptamer immobilization method. Additionally to the mentioned hindrances for

355

SPR measurement of macromolecular analytes (e. g. flow system or/and different weights of

356

interacting partners), the overall high densities of aptamers on sensor chip surfaces seem to

357

be responsible for further steric hindrances and limitations towards evaluation of whole

358

spore measurements. The utilization of biotin-streptavidin coupling and biotinylated

359

aptamers with an appropriate spacer (triethylene glycol, TEG) enable SPR measurements of

360

spore lysates by reducing density and increasing flexibility of formed aptamer layer by

361

concurrent reduction of target weight. This developed approach is completed by a suitable

362

regeneration procedure, which was accomplished by regeneration buffer 2. In conclusion, 16 ACS Paragon Plus Environment

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363

the usage of modified aptamers for immobilization and injection of spore lysate fulfill the

364

requirements for the determination of aptamer selectivity via SPR.

365

To compare the obtained SPR data of different aptamers and various endospores it was

366

necessary to immobilize equal amounts of streptavidin and aptamers in terms of RU signals

367

respectively ligand densities and inject similar spore concentrations. The spore suspensions

368

were diluted to a concentration of 106 cfu/mL and lysed according to the above mentioned

369

protocol for fragmentation of Alicyclobacillus spp. in the context of sensor chip preparation.

370

The lysate of Alicyclobacillus acidoterrestris spores (DSM 2498, wild type), which were used

371

as aptamer selection target (1/1, v/v), was employed as reference and set as 100 % aptamer

372

affinity to the target to compare various species and strains (exemplary SPR sensorgram

373

overlay in Figure 4).

374

The results of aptamer selectivity determination via SPR are stated in Table 4, all four

375

aptamers (AliApt 1, AliApt 3, AliApt 5, and AliApt 9) which have been immobilized show high

376

affinities towards various Alicyclobacillus spp. and support the implemented KD-

377

determinations. It should be noted, that the four aptamers also possess a high affinity (76 to

378

> 100 % relative to SELEX target Alicyclobacillus spp. spores (DSM 2498 and a wild type) to

379

two (AliApt 5 and AliApt 9) respectively three (AliApt 1 and AliApt 3) of the twelve different

380

Bacillus spp. spores. In general, it seems that the investigated aptamers are more affine to

381

spores of the genus Alicyclobacillus but also interacting with several Bacillus and Clostridium

382

spores (Table 4). The gained aptamer selectivity results via SPR were confirmed by

383

fluorometric binding assay using fluorescently labeled aptamers (data not shown).

384

An explanation could be that the spore coates of Alicyclobacillus, Bacillus, and Clostridium

385

are particularly similar and therefore the sensitivity of the generated aptamers is not

386

sufficient enough to distinguish between the investigated genera. Due to the partially high 17 ACS Paragon Plus Environment


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387

conformity in genome sequences of about 90 % between Alicyclobacillus and Bacillus it

388

might be reasonable that the spore surfaces are highly similar and therefore the generated

389

aptamers possess an affinity to homologous spore surface proteins.44,

390

evident (Table 4) that the obtained aptamers are more affine to spores of Alicyclobacillus as

391

of Bacillus and Clostridium and therefore usable for aptamer-based enrichment techniques.

392

An extension towards a highly specific detection system could be achieved by combination

393

with more specific molecular biological techniques, like real-time PCR or LAMP (loop-

394

mediated isothermal amplification).46

45

However, it is

395

Visualization of Binding-Interactions via Fluorescence Microscopy. Interactions between

396

generated aptamers and Alicyclobacillus spores were visualized using a fluorescence

397

microscope. In addition the orange juice simulating buffer containing Alicyclobacillus spores

398

only was considered as well and used as reference to exclude the intrinsic fluorescence of

399

spores. As shown in Figure 5 the spores exhibit a fluorescence signal after incubation with

400

fluorescently labeled aptamers, while spores by itself show no fluorescence signal under

401

chosen conditions. Thereby it was possible to visualize the interactions between the

402

generated aptamers and Alicyclobacillus spore coat via fluorescence microscopy.

403

During the presented work aptamers with an affinity to Alicyclobacillus spores were selected

404

by spore-SELEX. The aptamer selection process was carried out in orange juice simulating

405

buffer to obtain aptamers which are able to bind the mentioned spores under real

406

conditions. Furthermore the process was verified by various techniques (flow cytometric

407

binding assay, AGE, and radioactive binding assay). The gained aptamer sequences after 17

408

selection rounds were compared concerning secondary structures. For characterization

409

(determination of dissociations constants and aptamer selectivity) several SPR assays have

410

been developed by both immobilization of spore lysates respectively modified aptamers and 18 ACS Paragon Plus Environment

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411

were confirmed by fluorescence binding assay. Concerning KD values and selectivity suitable

412

aptamers for corresponding enrichment techniques were obtained. Furthermore the

413

interactions between intact Alicyclobacillus spore coat and gained aptamer mix were

414

visualized by fluorescence microscopy. To overcome microbiological plating methods the

415

gained aptamers could be integrated into aptamer-based techniques (e. g. magnetic

416

separation) for the enrichment of Alicycobacillus spores. Due to the lack of aptamer

417

selectivity regarding differentiation of fruit juice related microbiological contaminants the

418

combination with further molecular biological techniques, like real-time PCR or LAMP, is

419

necessary to increase sensitivity of the detection system.

420

In future the development and validation of an aptamer-based trapping and enrichment

421

technique and the adaption to real orange juice samples will be integrated and compared to

422

corresponding antibody-based systems. Furthermore the development of different aptamer-

423

based and rapid in-field applications like lateral flow dipsticks/devices (LFDs) should be

424

considered.47 Also the replacement of antibodies in well-established techniques like enzyme-

425

linked immunosorbent assay (ELISA) to develop a sensitive and easy applicable

426

Alicyclobacillus spores detection system is conceivable.

427

SUPPORTING INFORMATION

428

Radioactive binding assay (material and method respectively results), flow cytometric

429

binding assay (material and method respectively results), predicted secondary structures of

430

obtained aptamers (AliApt1 to AliApt10), overlays of several AliApt injection sensorgrams.

431

This material is available free of charge via the Internet at http://pubs.acs.org.

432 433 434 19 ACS Paragon Plus Environment


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435

FUNDING

436

This research project was supported by the German Ministry of Economics and Technology

437

(via AiF) and the FEI (Forschungskreis der Ernährungsindustrie e. V., Bonn, Germany). Project

438

AiF 17245 N.

439 440

ACKNOWLEDGEMENT

441

The authors gratefully acknowledge Daniel Schwark for his help in various parts of the

442

presented work, Dr. Burkhard Schütze (LADR GmbH, Geesthacht, Germany) for providing

443

various Alicyclobacillus spp. strains (spores and vegetative cells), and the Institute of

444

Biochemistry and Molecular Biology, University of Hamburg, for providing the technical

445

equipment and their support in several fields. We also thank Sven Malik from Sierra Sensors

446

GmbH, Hamburg, Germany for the support with the SPR evaluations. Furthermore we

447

acknowledge our project partners Ludwig-Maximilians-Universität München, Lehrstuhl für

448

Hygiene und Technologie der Milch (Oberschleißheim, Germany) and the Verband der

449

Deutschen Fruchtsaft-Industrie e.V. (Bonn, Germany) for support.

450


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LIST OF FIGURE CAPTIONS Figure 1: Schematic illustration of DNA aptamer selection for Alicyclobacillus spores by spore-SELEX in orange juice simulating buffer. Figure 2: Secondary structural features with corresponding dissociation constants of AliApt1, AliApt3, AliApt5, and AliApt9 as predicted using mfold V4.6. Figure 3: Overlay of AliApt3 sensorgrams with 360 - 840 pg immobilized spores fragments and several aptamer concentrations (A is 10 µM, B is 7.5 µM, C is 5 µM, and D is 2.5 µM). SPR measurements were carried out using orange juice simulating buffer (25 µL/min) to retain the secondary structures of aptamers. The double referenced data are evaluated with AnalyserR2 (Sierra Sensors GmbH, Hamburg, Germany) and Scrubber (BioLogic Software Pty Ltd, Campbell, Australia). Figure 4: Exemplary SPR sensorgram overlay of spore lysate injections of (A) A. acidoterrestris as reference (DSM 2498, wild type, 107 cfu/mL), (B) B. cereus (wild type, 107 cfu/mL), and (C) C. perfringens (wild type, 107 cfu/mL), with immobilized streptavidin and 5’-Biotin-TEG aptamer. SPR measurements were carried out using orange juice simulating buffer (25 µL/min) to retain the secondary structures of aptamers. Figure 5: Fluorescence microscopy recordings of A. acidoterrestris spores in orange juice simulating buffer as blank sample (1A to 1C with 40 x lens). Exemplary images of spores with prior incubation with a fluorescently labeled mix of obtained aptamers (2A to 2C with 40 x lens, 3A to 3C with 100 x lens). 1A to 3A show fluorescence images, 1B to 3B visual images, and 1C to 3C overlays of the fluorescence and visual images.



Page 28 of 37

FIGURES AND TABLES

Figure 1


Page 29 of 37


Figure 2



Page 30 of 37

Figure 3


Page 31 of 37


Figure 4



Page 32 of 37

Figure 5


Page 33 of 37


Table 1: Summary of SELEX conditions to increase stringency during selection rounds. SELEX round 1–4 5–6 7 8 9 10 11 12 – 13 14 – 15 16 – 17

Volume of Incubation Washing steps spore mix [µL] time [min] 20 60 1 20 40 1 20 35 1 20 30 2 20 25 2 20 20 2 15 15 2 15 15 3 10 15 3 10 10 3



Page 34 of 37

Table 2: Sequences and SPR estimated dissociation constants of aptamers for Alicyclobacillus

spores.

Aptamer

Aptamer sequence (5’-3’)

KD [nM]

AliApt1

CATCCGTCACACCTGCTCCATCCGTCACACCTGCTCACGTCATCCGTCA CACCTGCTCGGTGTTCGGTCCCGTATC

11.3

AliApt2

CATCCGTCACACCTGCTCCCATACCAGCCCCTGGTGTTGGCTCCCCGTA TCACCGCTCGGTGTTCGGTCCCGTATC

32.8

AliApt3

CATCCGTCACACCTGCTCCAGCGTGCGTCGACCCCGGACCCTGTCAGC CCCCCTCGCGGGTGTTCGGTCCCGTATC

2.7

AliApt4

CATCCGTCACACCTGCTCCACCCGTCACACTGGTGTTGGCTCCCGTATC ACCCGACTCGGTGTTCGGTCCCGTATC

37.6

AliApt5

CATCCGTCACACCTGCTCCCAGCGTGGCGTCGACCCGGACCCTGTCA GCCCCCTCGCGGGTGTTCGGTCCCGTATC

2.3

AliApt6

CATCCGTCACACCTGCTCCATCCGTCACACCTGCTCCCAGACCTGTCCT GCTCCCCTTGGTGTTCGGTCCCGTATC

33.0

AliApt7

CATCCGTCACACCTGCTCCCCACAACCGGTCCGGTGTTCGGTCCCCGT ATCACGCCTCGGTGTTCGGTCCCGTATC

149.1

AliApt8

CATCCGTCACACCTGCTCCCCCCTGGTGCATGGGTGTTCGGTCCCGTA TCACGCCTCGGTGTTCGGTCCCGTATC

34.4

AliApt9

CATCCGTCACACCTGCTCCACCCGTCACACCCATCCGTCACACCTGCTC CCCCCACTGGGTGTTCGGTCCCGTATC

16.0

AliApt10

CATCCGTCACACCTGCTCCCGTATCACCCCTCATCCGTCACACCCTGCT CCTACTGCGGGTGTTCGGTCCCGTATC

101.8


Page 35 of 37


Table 3: Different Alicyclobacillus, Bacillus, and Clostridium spores used for determination of

aptamer selectivity via SPR. Alicylobacillus spores

Bacillus spores

A. acidoterrestris (DSM 2498)

4x B. cereus (wild types)


B. weihenstepanensis (wild type)


B. thuringiensis (ATCC 10792)


B. thuringiensis (DSM 6029)

A. cycloheptanicus (DSM 4005)

B. licheniformis (DSM 13)


B. subtilis (DSM 2109)


B. coagulans (ATCC 7050)

A. acidocaldarius (DSM 446)

B. polymyxa (ATCC 10401)

A. herbarius (DSM 13609)

B. circulans (ATCC 9966)

A. acidiphilus (DSM 14558)

B. sphaericus (ATCC 245)

A. pomorum (DSM 14955) A. sacchari (DSM 17974) A. fastidiosus (DSM 17978)

Clostridium spores 2x C. perfringens (wild types)

34x undefined wild types



Page 36 of 37

Table 4: Summary of aptamer sensitivity of strains given in table 3. The genus Alicyclobacillus

is marked as A, Bacillus and Clostridium as B respectively C. The code (I-VI) displays the percentage of the ΔRU value related to the ΔRU value of the reference spore mix: (I) > 100 %, (II) 76-100%, (III) 51-75%, (IV) 26-50%, (V) 0-25%, and (VI) < 0 %.

I

II

III

IV

V

VI

Total

AliApt1 AliApt3 AliApt5 AliApt9 9A 11 A 7A 7A 3B 3B 2B 1B 0C 0C 0C 0C 6A 5A 3A 4A 0B 0B 0B 1B 0C 0C 0C 0C 3A 4A 0A 2A 0B 1B 0B 0B 0C 1C 0C 0C 14 A 13 A 10 A 1A 2B 2B 0B 1B 1C 1C 0C 0C 11 A 11 A 9A 7A 1B 2B 0B 0B 1C 0C 0C 0C 4A 3A 17 A 26 A 7B 5B 11 B 11 B 0C 0C 2C 1C 46 A 46 A 46 A 46 A 13 B 13 B 13 B 13 B 2C 2C 2C 2C


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TOC graphic


Food Sensing: Selection and Characterization of DNA Aptamers to

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