title: Selection of Single-Stranded DNA Molecular Recognition Elements against Exotoxin A Using a Novel Decoy-SELEX Method and Sensitive Detection of Exotoxin A in Human Serum.
abstract: Exotoxin A is one of the virulence factors of Pseudomonas aeruginosa, a bacterium that can cause infections resulting in adverse health outcomes and increased burden to health care systems. Current methods of diagnosing P. aeruginosa infections are time consuming and can require significant preparation of patient samples. This study utilized a novel variation of the Systematic Evolution of Ligand by Exponential Enrichment, Decoy-SELEX, to identify an Exotoxin A specific single-stranded DNA (ssDNA) molecular recognition element (MRE). Its emphasis is on increasing stringency in directing binding toward free target of interest and at the same time decreasing binding toward negative targets. A ssDNA MRE with specificity and affinity was identified after fourteen rounds of Decoy-SELEX. Utilizing surface plasmon resonance measurements, the determined equilibrium dissociation constant (Kd ) of the MRE is between 4.2 µM and 4.5 µM, and is highly selective for Exotoxin A over negative targets. A ssDNA MRE modified sandwich enzyme-linked immunosorbent assay (ELISA) has been developed and achieved sensitive detection of Exotoxin A at nanomolar concentrations in human serum. This study has demonstrated the proof-of-principle of using a ssDNA MRE as a clinical diagnostic tool.
authors.affiliation: Department of Pharmaceutical Sciences, West Virginia University, 1 Medical Center Drive, P.O. Box 9530, Morgantown, WV 20506, USA.
authors.forename: Ka Lok
authors.initials: KL
authors.lastname: Hong
authors.affiliation: School of Medicine, WV University, Morgantown, WV 20506, USA.
authors.forename: Kailey
authors.initials: K
authors.lastname: Yancey
authors.affiliation: Department of Medical Laboratory Science, West Virginia University, Morgantown, WV 20506, USA.
authors.forename: Luisa
authors.initials: L
authors.lastname: Battistella
authors.affiliation: Department of Pharmaceutical Sciences, West Virginia University, 1 Medical Center Drive, P.O. Box 9530, Morgantown, WV 20506, USA ; Molecular Pharmacology & Chemistry Program, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA.
authors.forename: Ryan M
authors.initials: RM
authors.lastname: Williams
authors.affiliation: Department of Pharmaceutical Sciences, West Virginia University, 1 Medical Center Drive, P.O. Box 9530, Morgantown, WV 20506, USA.
authors.forename: Katherine M
authors.initials: KM
authors.lastname: Hickey
authors.affiliation: Department of Pharmaceutical Sciences, West Virginia University, 1 Medical Center Drive, P.O. Box 9530, Morgantown, WV 20506, USA.
authors.forename: Chris D
authors.initials: CD
authors.lastname: Bostick
authors.affiliation: College of Pharmacy, Nova Southeastern University, Ft. Lauderdale, FL 33328, USA.
authors.forename: Peter M
authors.initials: PM
authors.lastname: Gannett
authors.affiliation: Department of Pharmaceutical Sciences, West Virginia University, 1 Medical Center Drive, P.O. Box 9530, Morgantown, WV 20506, USA.
authors.forename: Letha J
authors.initials: LJ
authors.lastname: Sooter
mesh_terms: D015415:Biomarkers / Q000097:blood; D001774:Blood Chemical Analysis / Q000379:methods; D004277:DNA, Single-Stranded / Q000097:blood* / Q000235:genetics; D005098:Exotoxins / Q000097:blood* / Q000235:genetics; D006801:Humans; D015203:Reproducibility of Results; D052156:SELEX Aptamer Technique / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D015415:Biomarkers; D004277:DNA, Single-Stranded; D005098:Exotoxins