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. 2023 Dec 8;22(1):e140505.
doi: 10.5812/ijpr-140505. eCollection 2023 Jan-Dec.

Screening and Identification of DNA Nanostructure Aptamer Using the SELEX Method for ‎Detection of Epsilon Toxin

Nafiseh Shafiei  1 , Hamideh Mahmoodzadeh Hosseini  2 , Jafar Amani  2 , Seyed Ali Mirhosseini  2 , Hanieh Jafary  1
Affiliations

Screening and Identification of DNA Nanostructure Aptamer Using the SELEX Method for ‎Detection of Epsilon Toxin

Nafiseh Shafiei et al. Iran J Pharm Res. .

Abstract

Background: Epsilon toxin (ETX), produced by Clostridium perfringens, is one of the most potent toxins known, with a lethal potency approaching that of botulinum neurotoxins. Epsilon toxin is responsible for enteritis. Therefore, the development of rapid and simple methods to detect ETX is imperative. Aptamers are single-stranded oligonucleotides that can bind tightly to specific target molecules with an affinity comparable to that of monoclonal antibodies (mAbs). DNA aptamers can serve as tools for the molecular identification of organisms, such as pathogen subspecies.

Objectives: This study aimed to isolate high-affinity single-stranded DNA (ssDNA) aptamers against ETX.

Methods: This study identified aptamers using the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) method, enzyme-linked apta-sorbent assay (ELASA), and surface plasmon resonance (SPR) to determine the affinity and specificity of the newly obtained aptamers targeting ETX.

Results: Several aptamers obtained through the SELEX process were studied. Among them, 2 aptamers, ETX clone 3 (ETX3; dissociation constant (Kd = 8.4 ± 2.4E-9M) and ETX11 (Kd = 6.3 ± 1.3E-9M) had favorable specificity for ETX. The limits of detection were 0.21 and 0.08 μg/mL for ETX3 and ETX11, respectively.‎.

Conclusions: The discovered aptamers can be used in various aptamer-based rapid diagnostic tests for the detection of ETX.

Keywords: Clostridium perfringens; DNA Aptamer; ELASA; Epsilon Toxin; SELEX; SPR.

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Figures

Figure 1.
Figure 1.. Asymmetric PCR amplification products of each SELEX round. M: 50 bp DNA ladder; 1 - 7: SELEX round numbers
Figure 2.
Figure 2.. Binding assay of aptamers against the ETX protein in ELASA. The binding affinity of the aptamers for rounds 1, 3, 5, and 7 of SELEX was evaluated and compared with 3 wells, one containing protein, one containing aptamer, and one without aptamer, and protein as negative control groups. Round 7 showed the highest binding affinity
Figure 3.
Figure 3.. Selection and characterization of the best sequence of aptamers. A, Agarose gel electrophoresis of colony polymerase chain reaction (PCR) products from clones 1 - 11. Clones 3 and 11 were selected and confirmed using colony PCR. M: 50 bp DNA ladder; 1 - 11: Clone 1 to 11; B, enzyme-linked apta-sorbent assay (ELASA) analysis of the 2 cloned aptamer candidates that bind epsilon toxin (ETX). Each of the 2 aptamer sequences is named ETX3 and 11, with optical density (ODs) of 0.9 and 0.6, respectively; C, The secondary structures of aptamer ETX3 and ETX11 were predicted using the UNAFold online program according to the free energy minimization algorithm.
Figure 4.
Figure 4.. Aptamer sensitivity and optimization using the enzyme-linked apta-sorbent assay method. A, different concentrations of the epsilon protein against the concentrations of epsilon toxin clone 3 (ETX3) and ETX11 aptamers; B, different concentrations of ETX3 and ETX11 aptamers against the concentration of the epsilon protein
Figure 5.
Figure 5.. Surface plasmon resonance (SPR) analysis of aptamers for Kd determination. A range of aptamer concentrations (1.5, 15, 150, and 1500 nM) prepared in 10 mM PBST (PH = 7.4) was passed over the surface containing immobilized ETX (50 µg). A, the sensorgram of SPR was used to evaluate the binding affinity between the epsilon protein and the ETX3 aptamer; B, the sensorgram of SPR to evaluate the binding affinity between the epsilon protein and ETX11 aptamer
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