Rapid isolation of bacteria-specific aptamers with a non-SELEX-based method

Rapid isolation of bacteria-specific aptamers with a non-SELEX-based method

Journal Pre-proof Rapid isolation of bacteria-specific aptamers with a non-SELEX-based method Hye Ri Kim, Min Yong Song, Byoung Chan Kim PII: S0003-2...

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Journal Pre-proof Rapid isolation of bacteria-specific aptamers with a non-SELEX-based method Hye Ri Kim, Min Yong Song, Byoung Chan Kim PII:

S0003-2697(19)30976-5

DOI:

https://doi.org/10.1016/j.ab.2019.113542

Reference:

YABIO 113542

To appear in:

Analytical Biochemistry

Received Date: 4 October 2019 Revised Date:

9 December 2019

Accepted Date: 10 December 2019

Please cite this article as: H.R. Kim, M.Y. Song, B. Chan Kim, Rapid isolation of bacteria-specific aptamers with a non-SELEX-based method, Analytical Biochemistry (2020), doi: https://doi.org/10.1016/ j.ab.2019.113542. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc.

Author Statement Hye Ri Kim: Validation, Investigation, Data curation, Writing-Original draft, WritingReviewing and Editing; Min Yong Song: Validation, Investigation, Data curation, WritingOriginal draft, Writing- Reviewing and Editing; Byoung Chan Kim: Conceptualization, Methodology, Formal analysis, Writing-Original draft, Writing- Reviewing and Editing

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Rapid isolation of bacteria-specific aptamers with a non-SELEX-

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based method

3 Hye Ri Kima,b,1, Min Yong Songc,1, and Byoung Chan Kima,b,*

4 5 6

a

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Technology (KIST), Hwarangno 14-gil 5, Seongbuk-gu, Seoul 02792, Republic of Korea

8

b

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Technology (UST), Hwarangno 14-gil 5, Seongbuk-gu, Seoul 02792, Republic of Korea

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c

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;

Center for Environment, Health and Welfare Research, Korea Institute of Science and

Division of Energy and Environment Technology, KIST School, University of Science and

Seoul Institute of Technology, Maebongsan-ro 37, Mapo-gu, Seoul 03909, Republic of Korea

13 14 15 16

1

17

* Corresponding author

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Byoung Chan Kim, Ph.D.

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Tel: + 82 2 958 5877

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Fax: + 82 2 958 5805

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E-mail address: [email protected] (B.C. Kim)

Authors contributed equally

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For Technical notes (Analytical Biochemistry)

1

Abstract

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Usually, isolation of bacteria-specific aptamers by SELEX is a time-consuming

3

process due to the required repeated rounds of binding, separation, and amplification of

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the probes to target bacteria. Here, we show that it is possible to isolate bacteria-specific

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DNA aptamers omitting the repeated rounds of binding incubation, separation, and

6

amplification that are indispensable for SELEX. The serial removal of unbound DNAs

7

to the bacterial cells from an initial mixture of bacteria and DNA libraries through serial

8

centrifugation, one-time separation, and further one-time amplification of DNA bound

9

to the target bacterial cells applied in this non-SELEX-based method allows successful

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aptamer isolation.

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Key Words: centrifugation-based partitioning; rapid isolation; bacteria-specific

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aptamer; non-SELEX-based method; Escherichia coli

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1

1. Introduction

2 3

Microorganisms represent the most abundant and diverse species on Earth [1].

4

Microbial communities contain hundreds or thousands of species, most of which

5

cannot be detected, although progress has been made in the development of

6

microscopic instruments, cultivation techniques, and molecular-based diagnostics

7

using monoclonal and polyclonal antibodies or nucleic acids. Antibodies have

8

become indispensable tools in diagnostic analyses and tests that are routinely

9

used for specific detection and precise quantification of target microorganisms [2-

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4]. However, considering the number of existing microorganisms, the production

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of antibodies for this purpose is substantially limited. Antibody production

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usually starts with the identification of antibodies using animals and hybridomas.

13

Additionally, the processes employed in the production of monoclonal and

14

polyclonal antibodies are expensive and depend on intensive skilled labor, being

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practically impossible to produce antibodies against all microorganisms. In terms

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of developing specific receptors for all microorganisms, alternative techniques

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are highly demanded.

18 19

Bacteria-specific aptamers, the nucleic acids that bind to target bacterial cells with

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high affinity and specificity, can be screened by the SELEX (Systematic Evolution of

21

Ligands by Exponential Enrichment) process and are considered as an alternative

22

approach for the production of target cell-specific binding receptors due to its low cost

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and in vitro-based production [2, 5-7]. Because of its easy accessibility compared to

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antibodies, many cell (animal, human, and bacterial cells)-specific receptors (aptamers)

3

1

were developed through the Cell-SELEX technology and reported [5, 8]. Cell-SELEX

2

usually includes 7–30 cycles of incubation, separation, and amplification of probes from

3

random nucleic acid libraries to enrich the oligonucleotides that are bound to the target

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cells. Subsequently, enriched oligonucleotide pools from PCR amplification are cloned

5

and sequenced [5, 9, 10]. Because the enrichment procedure includes repetitive steps of

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probe incubation with target bacterial cells, separation of bound probes from target

7

bacterial cells, and amplification, the final aptamers become rigid in terms of affinity

8

and specificity to the targets. It makes sense that repeated enrichment steps are essential

9

to obtain high-affinity aptamers. However, although numerous studies demonstrated

10

that the SELEX process generates better aptamers to bacterial cells, the repetitive

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enrichment steps also elevate the cost and time associated to this process and require

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extremely large amounts of the target cells for aptamer isolation [11-14]. To overcome

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the drawbacks of SELEX, new aptamer selection processes have been introduced to

14

reduce some steps of SELEX. Nitsche and colleagues have suggested a one-step method

15

for the isolation of aptamers (MonoLEX) against orthopoxvirus [9]. That method

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comprised chemical coupling of virus particles to the affinity column, incubation with a

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random DNA library (RDL), extensive washing to elute non-binding oligonucleotides,

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physical segmentation of the affinity column, extraction of oligonucleotides from each

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column segment, and amplification of the oligonucleotides extracted from each column

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segment using PCR to obtain high affinity aptamers. Similarly, Berezovski and

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colleagues reported the use of another non-SELEX-based method (non-equilibrium

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capillary electrophoresis of equilibrium mixtures (NECEEM)-based partitioning) to

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obtain high-affinity aptamers against the h-Ras protein [15]. The NECEEM-based

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method can isolate high-affinity aptamers by the repetitive partitioning of

4

1

oligonucleotides against the target without a PCR amplification step. Both methods

2

have shown that it was possible to isolate high-affinity aptamers against viruses or

3

proteins without enrichment or amplification steps. Unlike targets such as virus particles

4

or proteins, bacterial cells can be easily separated from the aqueous reaction mixture by

5

centrifugation under the appropriate centrifugal forces. Indeed, Hamula and colleagues

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reported a modified cell-based SELEX method against live bacterial cells [13].

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Following centrifugation, the cells were eluted from cell-bound aptamers, and fractions

8

in the isolated supernatant were amplified by PCR for the next round of selection.

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However, this method still requires repetitive elution and PCR amplification for a total

10

of 8 rounds of the SELEX process.

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Unlike these methods, we propose a rapid method to isolate bacterial cell-specific

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DNA aptamers that does not require repeated rounds of elution and amplification of

14

bound probes. Therefore, the method suggested in this study is not called SELEX. We

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used Escherichia coli as a model to validate the method suggested in this study. The

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repetitive centrifugation-based partitioning between bacterial cells bound to DNA and

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unbound DNA enables the rejection of unbound DNA through sequential

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centrifugations of the initial mixture of target bacterial cells and RDL pools. The weakly

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bound DNAs are rejected during the excessive and sequential partitioning by

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centrifugation. Finally, in this method, the DNA pools bound to cells (E. coli) are

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amplified only once prior to cloning, while SELEX needs repetitive rounds of binding,

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elution, and amplification. The affinity of the aptamers isolated through this method

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was compared to that of the aptamers isolated by the SELEX method [11].

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(Here figure 1)

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1 2

2. Materials and Methods

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2.1. Bacterial strains and culture media

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E. coli (KCTC 2571), Bacillus subtilis (KCTC 1022), Klebsiella pneumonia (KCTC

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2208), Citrobacter freundii (KCTC 2006), Enterobacter aerogenes (KCTC 2190), and

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Staphylococcus epidermidis (KCTC 1917) were obtained from the KCTC (Korean

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Collection for Type Culture, Daejeon, Republic of Korea). Nutrient broth and nutrient

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agar were purchased from Becton, Dickinson and Company (Franklin Lakes, NJ, USA).

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E. coli, S. epidermidis, and K. pneumonia were cultivated at 37 °C. E. aerogenes, C.

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freundii, and B. subtilis were cultivated at 30 °C.

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2.2. RDL and primers

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An RDL (100 µM) and primers (10 µM) were purchased from GenoTech (Daejeon,

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Republic of Korea). The single-stranded RDL consisted of 45 random nucleotides

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flanked by two different sequences at the 3′ and 5′ ends (88-mer, 5′-GCA ATG GTA

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CGG TAC TTC C-N45-CAA AAG TGC ACG CTA CTT TGC TAA-3′). The sequences

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of forward and reverse primers for PCR were 5′-GCA ATG GTA CGG TAC TTC C-3′

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(19-mer) and 5′-TTA GCA AAG TAG CGT GCA CTT TTG-3′ (24-mer), respectively.

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2.3. Centrifugation-based partitioning selection process for the isolation of ssDNA

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1

aptamers

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The target E. coli cells were cultured in nutrient broth to reach the concentration of

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approximately 1 × 108 CFU mL-1. The cells (107 CFU) were washed three times with 1×

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phosphate-buffered saline (PBS, pH 7.0) by centrifugation (13,000 rpm) at 25 °C for 10

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min. The RDL was heated at 95 °C for 5 min and cooled down immediately on ice for

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10 min before mixing it with the cells. For aptamer selection, the denatured RDL (1.8

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nmol) was incubated with 107 E. coli cells using a binding buffer (1× PBS, 0.1 mg mL-1

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salmon sperm DNA, 1% bovine serum albumin, and 0.05% Tween-20) in Thermomixer

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(Eppendorf, Hamburg, Germany) with constant shaking (1,000 rpm) at room

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temperature for 1 h. The mixture was then centrifuged (13,000 rpm) for 5 min, and the

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supernatant was discarded to remove oligonucleotides and other ingredients in a binding

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buffer that did not bind to E. coli. To remove the oligonucleotides weakly bound to E.

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coli cells, the latter were resuspended using 1× PBS (washing buffer, 1 mL, pH 7.0) and

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then vortexed at room temperature for 5 min (Vortex-Genie 2 Mixer, Scientific

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Industries, Bohemia, NY, USA). The cell suspension was recovered by centrifugation

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(13,000 rpm) at room temperature for 10 min, and then the supernatant was discarded.

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The amount of bound DNAs was calculated by measuring the concentration of DNAs

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washed out in the supernatant after centrifugation using an ND 1000 Spectrophotometer

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(NanoDrop, Thermo Fisher Scientific, Wilmington, DE, USA). This centrifugation-

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based partitioning process, including centrifugation, removal of the supernatant,

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resuspension, and vortexing, was repeated 10 times sequentially. After 10 cycles, the

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negative selection was performed using a mixture of other bacterial species (B. subtilis,

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K. pneumonia, C. freundii, E. aerogenes, and S. epidermidis) that has 107 cells

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1

approximately in 1 X PBS (a number of each bacterium is 2.0 X 106 CFU cells

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approximately). For the negative selection, we eluted the bound DNA from E. coli cells.

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In detail, E. coli cells were resuspended in 100 µL of autoclaved DNase-free water,

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heated at 95 °C for 10 min, and then centrifuged (13,000 rpm) for 10 min at room

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temperature. We harvested the supernatant and then mixed it with the negative selection

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pools (1 mL in 1 X PBS). The mixture was incubated for 1 hr at 25 °C under shaking

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condition (250 rpm). The mixture was centrifuged (13,000 rpm) for 10 min to remove

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the negative selection pools and then the only supernatant was harvested. With this

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supernatant and E. coli cells, we performed additional 10 times centrifugation-

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partitioning in 1 X PBS condition. After final partitioning, we eluted the bound

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oligonucleotides or DNAs from E. coli through the same method described previously.

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The supernatant eluted was filtered using centrifugal filter units (Amicon Ultra-0.5 mL

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50 K centrifugal filter units, Millipore Ireland Ltd., Cork, Ireland) to get rid of

14

contaminants such as cells, BSA, or salmon sperm DNA and obtain the flowthrough

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fraction containing oligonucleotides that may have affinity to the targets. The purified

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oligonucleotides were chilled at 4 °C and then diluted in distilled water and PCR

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amplified to double-stranded DNA (dsDNA) using forward and reverse primers for

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cloning.

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2.4. Cloning and sequencing

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The amplified dsDNAs were cloned using a TOPO TA Cloning Kit (Invitrogen,

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Carlsbad, CA, USA) and transformed into E. coli DH10BTM cells (Invitrogen, Carlsbad,

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CA, USA). Cells were grown in agar plate (100 mM of IPTG, 40 mg/mL of X-gaL, and

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1

50 µg/mL ampicillin) overnight at 37 °C. After that, we picked white colonies, grew

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them in LB medium (50 µg/mL ampicillin) overnight at 37 °C, and then extracted

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plasmid DNA using a Miniprep Kit (Qiagen, Mississauga, ON, Canada). The sequence

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of the inserted dsDNA was analyzed by GenoTech (Daejeon, Republic of Korea). The

5

secondary structures of several selected aptamers were predicted by free energy

6

minimization algorithm using the Internet tool Mfold [16].

7 8

2.5. Affinity and selectivity test

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The affinity of the isolated aptamers was investigated by its binding to target E.

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coli cells. The cells (~ 107 cells suspended in 1× PBS (100 µL)) were incubated with

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various concentrations (0, 5, 12.5, 25, 50, 125, and 250 nM in the final binding reaction)

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of 3′-FAM dye-labeled aptamers (in 1× PBS (100 µL)) in a Thermomixer (Eppendorf)

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with constant shaking (1,000 rpm) at room temperature for 1 h. For the selectivity test,

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the binding of aptamers to different species of bacteria (B. subtilis, K. pneumonia, C.

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freundii, E. aerogenes, or S. epidermidis) was assessed using the same conditions

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described above with FAM dye-labeled aptamers at a final concentration of 250 nM. In

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all tests, we centrifuged the mixture of cells and FAM dye-labeled aptamers under

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13,000 rpm for 10 min at room temperature to get rid of unbound FAM dye-labeled

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aptamers. Finally, the fluorescence intensity of cells bound to 3′-FAM dye-labeled

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aptamers was measured using a micro-fluorospectrometer (NanoDrop-3300, Thermo

22

Fisher Scientific). To estimate the Kd (dissociation constant) value, fluorescence

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intensity values were plotted versus the respective concentrations of added aptamer and

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the relationship was fitted by non-linear regression (ligand-binding/one-site saturation

9

1

function)_using Sigmaplot 10.0 software (Systat Software, Inc., San Jose, CA, USA).

2 3

(Here figure 2)

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3. Results and Discussion

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3.1. Centrifugation-based partitioning method for the isolation of aptamers

8 9

Aptamers are usually isolated from RDLs using the SELEX method. The Cell-

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SELEX method is especially suitable for the selection of aptamers that are specific to

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living mammalian or bacterial cells [17-19]. Usually, SELEX includes multiple rounds

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of enrichment steps involving separation of unbound oligonucleotides from the targets

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and amplification of bound oligonucleotides by PCR for the next enrichment round [6,

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20, 21]. Although these enrichment steps are helpful for the isolation of aptamers that

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are highly specific to the targets, they are part of a time-consuming process that requires

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specialized instruments and skills [22, 23]. In the cases of infections with emerging

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bacterial pathogens, the latter have to be detected and treated urgently; therefore,

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specific diagnostic tools and therapeutic agents are required [3, 24, 25]. Considering

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this, oligonucleotide aptamers are suitable tools as their preparation is more rapid

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compared to that of antibodies. Here, we introduce a new centrifugation-based

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partitioning method for the isolation of bacterial cell-specific aptamers, which, unlike

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the conventional SELEX method, does not contain an enrichment step, so the total time

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for the isolation process is shorter (Figure 1). We isolated E. coli-specific DNA

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aptamers using the centrifugation-based partitioning method as a model case. If the

10

1

number of centrifugation-based partitioning cycles is increased infinitely, the

2

oligonucleotides bound to the targets after final centrifugation-based partitioning can be

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presumed to have high binding affinity to the targets. First, E. coli cells were incubated

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with an RDL. Oligonucleotides bound to E. coli were recovered, whereas unbound or

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weakly bound oligonucleotides were removed by repetitive centrifugation-based

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partitioning steps. We performed 20 cycles of centrifugation-based partitioning because

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the unbound DNAs were not measurable after these 20 cycles, and obtained the final

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oligonucleotides bound to E. coli. The amount of total DNA (oligonucleotides from the

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RDL and salmon sperm DNA from the binding buffer) that bound to the target cells

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became gradually lower at each partitioning (Figure 2). This indicates that unbound or

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weakly bound oligonucleotides were sequentially removed by repetitive partitioning and

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bound oligonucleotides remained tightly associated to the surface of E. coli. The

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repeated partitioning process based on centrifugation and washing can help to obtain

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aptamers with high binding affinity to the targets because oligonucleotides that have

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low or no affinity will be discarded during the partitioning. After 10 partitioning cycles,

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negative selection was performed to improve the specificity and selectivity of the

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aptamers to the target cells using a mixture of other bacterial cells (B. subtilis, K.

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pneumonia, C. freundii, E. aerogenes, and S. epidermidis). After 20 cycles of

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centrifugation-based partitioning, the isolated oligonucleotides were amplified once and

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cloned for the determination of their sequences, whereas SELEX needs several iterative

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PCR steps and denaturation of dsDNA into ssDNA for the sequential enrichment round

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before sequence identification. A total of 10 different ssDNA sequences were identified

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(Table S1) among 45 plasmids that were extracted from white colonies after cloning.

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Usually, after SELEX, aptamer candidate sequences show highly conserved regions

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1

among them because the enrichment step generates the same sequences. In contrast, our

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centrifugation-based partitioning method yielded candidates with unique sequences, as

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there was no enrichment step and PCR was performed only once. The 20-cycle

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partitioning steps and one-time PCR, excluding cloning, took only approximately 7 h

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from the first binding between RDL and targets, although we still need to further

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optimize the number of partitioning steps.

7 8

(Here figure 3)

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3.2. Determination of affinity and investigation of selectivity of the obtained aptamers

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The 10 ssDNA sequences (Table S1) were inferred by Mfold to have definite

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stem-loop structures based on the surface free energy minimization algorithm.

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Among them, we selected three candidate sequences (20-5, 20-7, and 20-10) that

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have more than five definite stem-loops (Figure S1). These candidates were

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chosen and examined for their affinity to E. coli because previous studies have

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shown that stem-loop structures have an important role in binding to target cells

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[26]. The 3′-FAM dye-labeled sequences were used to perform the binding

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affinity assay after incubation and washing with E. coli. Figure 3 (A), (B), and

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(C) shows a representative binding saturation curve of each sequence to E. coli by

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fluorescence analysis. The dissociation constants (Kd) of the three sequences, 20-

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5, 20-7, and 20-10, were estimated to be 3.9 nM, 8.0 nM, and 10.1 nM,

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respectively. These Kd values were comparable to those of the aptamers (12.4 to

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25.2 nM) that were previously isolated by the SELEX method in our laboratory

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1

[27]. The selectivity of the three 3′-FAM dye-labeled aptamers (20-5, 20-7, and

2

20-10) was examined using the same concentration (250 nM) with target (E. coli)

3

and non-target (B. subtilis, K. pneumonia, C. freundii, E. aerogenes, and S.

4

epidermidis) bacterial cells. The latter were included as a negative selection

5

mixture pool. Figure 3 (D) shows distinct degrees of binding of these three

6

aptamers to E. coli, whereas the degree of binding to non-target bacteria was

7

below ~25% of that to target E. coli. Based on the affinity and selectivity tests,

8

the aptamers isolated via centrifugation-based partitioning had similar properties

9

to those isolated by the SELEX method and, therefore, may be interchangeable

10

for various purposes.

11 12

In this study, we showed that the centrifugation-based partitioning method

13

allows obtaining aptamers in a simple and efficient manner that reduces the

14

number of repetitive separation steps for the conversion of dsDNAs to

15

oligonucleotides after enrichment. It also does not require expensive instruments,

16

extensive and time-consuming labor, or a large amount of target. The results of

17

this study suggest that centrifugation-based partitioning is a promising method for

18

the isolation of aptamers against bacterial cells as it can be performed much faster

19

compared to SELEX-based aptamer isolation. Also, we think that this method can

20

be general for the other targets such as proteins or chemicals if they are immobilized to

21

the solid matrix appropriately that can be separable easily by using centrifugation.

22 23

4. Conclusions

13

1 2

The centrifugation-based partitioning method allowed isolation of aptamers specific

3

to E. coli cells with dissociation constants (in the nanomolar range) comparable to those

4

of aptamers obtained by the SELEX method. The centrifugation-based partitioning

5

method is rapid and simple compared to the SELEX method due to a lower number of

6

enrichment steps, allowing the entire process before the cloning step to be completed

7

within ~7 h. Although we only report the generation of aptamers against E. coli cells,

8

this method can be readily applied to obtain aptamers against other cells.

9 10

Acknowledgement

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This work was finally supported by the Korea Institute of Science and Technology (KIST) Institutional Research Program.

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[26] L. GhisolfiNieto, G. Joseph, F. PuvionDutilleul, F. Amalric, P. Bouvet, Nucleolin is a sequence-specific RNA-binding protein: Characterization of targets on preribosomal RNA, J. Mol. Biol., 260 (1996) 34-53. [27] Y.S. Kim, M.Y. Song, J. Jurng, B.C. Kim, Isolation and characterization of DNA aptamers against Escherichia coli using a bacterial cell-systematic evolution of ligands by exponential enrichment approach, Anal. Biochem., 436 (2013) 22-28.

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List of figures

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Figure 1. Schematic representation of the centrifugation-based partitioning process in

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comparison to Cell-SELEX of ssDNA aptamers

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Figure 2. The amount of total bound DNA calculated in each partitioning round

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Figure 3. Binding saturation curve of aptamers (A) 20-5, (B) 20-7, and (C) 20-10 to

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target Escherichia coli, and (D) binding intensity of 20-5, 20-7, and 20-10 to non-target

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bacteria (Bacillus subtilis, Klebsiella pneumonia, Citrobacter freundii, Enterobacter

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aerogenes, and Staphylococcus epidermidis)

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Figure 1.

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Figure 2.

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Figure 3.

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Highlights

Non-SELEX-based method for the isolation of aptamers to bacterial cells was proposed. Centrifugation-based partitioning method can reduce the time remarkably to isolate aptamers to target bacteria. The aptamers isolated by centrifugation-based partitioning method were comparable to the aptamers isolated by SELEX method.