In vitro selection of single-stranded DNA molecular recognition elements against S. aureus alpha toxin and sensitive detection in human serum

Abstract

Alpha toxin is one of the major virulence factors secreted by Staphylococcus aureus, a bacterium that is responsible for a wide variety of infections in both community and hospital settings. Due to the prevalence of S. aureus related infections and the emergence of methicillin-resistant S. aureus, rapid and accurate diagnosis of S. aureus infections is crucial in benefiting patient health outcomes. In this study, a rigorous Systematic Evolution of Ligands by Exponential Enrichment (SELEX) variant previously developed by our laboratory was utilized to select a single-stranded DNA molecular recognition element (MRE) targeting alpha toxin with high affinity and specificity. At the end of the 12-round selection, the selected MRE had an equilibrium dissociation constant (Kd) of 93.7 ± 7.0 nM. Additionally, a modified sandwich enzyme-linked immunosorbent assay (ELISA) was developed by using the selected ssDNA MRE as the toxin-capturing element and a sensitive detection of 200 nM alpha toxin in undiluted human serum samples was achieved.

Figure 1

Illustration of the Systematic Evolution of Ligands by EXponential enrichment (SELEX) process. A random library consisting of 10¹⁵ ssDNA molecules (each with a different nucleotide sequence, indicated by different shapes) were incubated with the target alpha toxin. Those DNA that bound to the target were amplified and then incubated with negative targets. Those DNA that do not bind to negative targets are amplified and subjected to further rounds of in vitro selection.

Figure 2

Sequence and secondary structure of R12.06 ssDNA MRE. Hydrogen bonds are indicated in red between GC base pairs and in black between AT base pairs. (A) ssDNA sequence of alpha toxin MRE R12.06; and (B) Mfold prediction of R12.06 secondary structure [24].

Figure 3

Illustration of the ssDNA MRE modified ELISA assay. The ssDNA MRE was used as the capturing element in the sandwich ELISA assay. The curved blue line attached to NH₂ represents the 5' amino modified MRE. The purple irregular shape represents alpha toxin. The red “Y” shape represents the primary antibody against alpha toxin. The grey oval represents horseradish peroxidase (HRP) conjugated to a secondary antibody (pink). The light green circle represents the substrate of HRP and the dark green circle represents the metabolite of HRP, which is detected with absorbance measurements.

Figure 4

Detection of alpha toxin in modified ELISA assay. Data represent one modified sandwich ELISA with absorbance measured at 410 nm. Absorbance levels are subtracted from background levels of blank wells without immobilized DNA. (A) Statistical significance levels with respect to DNA with buffer background of p < 0.001 are designated by *; and (B) Statistical significance levels with respect to human serum background of p < 0.001 are designated by *. Buffer: 1× selection buffer. Error bars are representative of ±1× standard deviations.

Figure 5

Structures of targets used in the SELEX scheme and cross binding assays. (A) Ribbon structure of the target of interest, alpha toxin (PDB 3ANZ, 33 kDa) [44]; (B–E) Ribbon structure of exotoxin A (PDB 1IKQ, 66 kDa) [45], bovine serum albumin (PDB 4F5S, 66.5 kDa) [46], cholera toxin (PDB 2A5D, 84 kDa) [47], and toxin B (PDB2BVM, 270 kDa) [48], used in negative rounds of selection and cross binding assays.