title: Selection of DNA aptamers against uropathogenic Escherichia coli NSM59 by quantitative PCR controlled Cell-SELEX.
abstract: In order to better control nosocomial infections, and facilitate the most prudent and effective use of antibiotics, improved strategies for the rapid detection and identification of problematic bacterial pathogens are required. DNA aptamers have much potential in the development of diagnostic assays and biosensors to address this important healthcare need, but further development of aptamers targeting common pathogens, and the strategies used to obtain specific aptamers are required. Here we demonstrate the application of a quantitative PCR (qPCR) controlled Cell-SELEX process, coupled with downstream secondary-conformation-based aptamer profiling. We used this approach to identify and select DNA aptamers targeted against uropathogenic Escherichia coli, for which specific aptamers are currently lacking, despite the prevalence of these infections. The use of qPCR to monitor the Cell-SELEX process permitted a minimal number of SELEX cycles to be employed, as well as the cycle-by-cycle optimisation of standard PCR amplification of recovered aptamer pools at each round. Identification of useful aptamer candidates was also facilitated by profiling of secondary conformations and selection based on putative aptamer secondary structure. One aptamer selected this way (designated EcA5-27), displaying a guanine-quadruplex sequence motif, was shown to have high affinity and specificity for target cells, and the potential to discriminate between distinct strains of E. coli, highlighting the possibility for development of aptamers selectively recognising pathogenic strains. Overall, the identified aptamers hold much potential for the development of rapid diagnostic assays for nosocomial urinary tract infections caused by E. coli.
authors.affiliation: Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan. Electronic address: 50012831103@st.tuat.ac.jp.
authors.forename: Nasa
authors.initials: N
authors.lastname: Savory
authors.affiliation: School of Pharmacy and Biomolecular Sciences, University of Brighton, Huxley Building, Lewes Road, Brighton, East Sussex, BN2 4GJ, United Kingdom. Electronic address: J.Nzakizwanayo@brighton.ac.uk.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Nzakizwanayo
authors.affiliation: Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan. Electronic address: abekou@cc.tuat.ac.jp.
authors.forename: Koichi
authors.initials: K
authors.lastname: Abe
authors.affiliation: School of Bioscience and Biotechnology, Tokyo University of Technology, 1404-1 Katakuramachi, Hachioji, Tokyo 192-0982, Japan. Electronic address: yoshidawtr@stf.teu.ac.jp.
authors.forename: Wataru
authors.initials: W
authors.lastname: Yoshida
authors.affiliation: Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan. Electronic address: stefano@cc.tuat.ac.jp.
authors.forename: Stefano
authors.initials: S
authors.lastname: Ferri
authors.affiliation: School of Pharmacy and Biomolecular Sciences, University of Brighton, Huxley Building, Lewes Road, Brighton, East Sussex, BN2 4GJ, United Kingdom. Electronic address: C.G.Dedi@brighton.ac.uk.
authors.forename: Cinzia
authors.initials: C
authors.lastname: Dedi
authors.affiliation: School of Pharmacy and Biomolecular Sciences, University of Brighton, Huxley Building, Lewes Road, Brighton, East Sussex, BN2 4GJ, United Kingdom; Queen Victoria Hospital NHS Foundation Trust, Holtye Road, East Grinstead, West Sussex, RD19 3DZ, United Kingdom. Electronic address: B.V.Jones@brighton.ac.uk.
authors.forename: Brian V
authors.initials: BV
authors.lastname: Jones
authors.affiliation: Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan. Electronic address: ikebu@cc.tuat.ac.jp.
authors.forename: Kazunori
authors.initials: K
authors.lastname: Ikebukuro
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D004927:Escherichia coli Infections / Q000382:microbiology*; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction; D052156:SELEX Aptamer Technique / Q000295:instrumentation / Q000379:methods*; D056689:Uropathogenic Escherichia coli / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D052157:Aptamers, Nucleotide
keywords: Aptamers; Cell-SELEX; Escherichia coli; Nosocomial infections; Urinary tract infections