title: Application of locked nucleic acid-based probes in fluorescence in situ hybridization.
abstract: Fluorescence in situ hybridization (FISH) employing nucleic acid mimics as probes is becoming an emerging molecular tool in the microbiology area for the detection and visualization of microorganisms. However, the impact that locked nucleic acid (LNA) and 2'-O-methyl (2'-OMe) RNA modifications have on the probe that is targeting microorganisms is unknown. In this study, the melting and hybridization efficiency properties of 18 different probes in regards to their use in FISH for the detection of the 16S rRNA of Helicobacter pylori were compared. For the same sequence and target, probe length and the type of nucleic acid mimics used as mixmers in LNA-based probes strongly influence the efficiency of detection. LNA probes with 10 to 15 mers showed the highest efficiency. Additionally, the combination of 2'-OMe RNA with LNA allowed an increase on the fluorescence intensities of the probes. Overall, these results have significant implications for the design and applications of LNA probes for the detection of microorganisms.
authors.affiliation: LEPABE, Laboratory for Process Engineering, Environment, Biotechnology and Energy, Department of Chemical Engineering, Faculty of Engineering, University of Porto, Porto, Portugal.|i3S, Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal.|IPATIMUP, Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal.|Nucleic Acid Center, Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Odense M, Denmark.|ICBAS, Institute of Biomedical Sciences Abel Salazar, University of Porto, Porto, Portugal.|Epithelial Cell Biology Group; Cancer Cell Biology Program, Spanish National Cancer Research Centre, Madrid, Spain.
authors.forename: Sílvia
authors.initials: S
authors.lastname: Fontenete
authors.affiliation: LEPABE, Laboratory for Process Engineering, Environment, Biotechnology and Energy, Department of Chemical Engineering, Faculty of Engineering, University of Porto, Porto, Portugal.
authors.forename: Daniel
authors.initials: D
authors.lastname: Carvalho
authors.affiliation: LEPABE, Laboratory for Process Engineering, Environment, Biotechnology and Energy, Department of Chemical Engineering, Faculty of Engineering, University of Porto, Porto, Portugal.|i3S, Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal.|IPATIMUP, Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal.|Nucleic Acid Center, Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Odense M, Denmark.
authors.forename: Nuno
authors.initials: N
authors.lastname: Guimarães
authors.affiliation: i3S, Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal.|ICBAS, Institute of Biomedical Sciences Abel Salazar, University of Porto, Porto, Portugal.|IBMC, Institute for Molecular Biology and Cell Biology, Porto, Portugal.
authors.forename: Pedro
authors.initials: P
authors.lastname: Madureira
authors.affiliation: i3S, Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal.|IPATIMUP, Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal.|FMUP, Faculty of Medicine of the University of Porto University, Porto, Portugal.
authors.forename: Céu
authors.initials: C
authors.lastname: Figueiredo
authors.affiliation: Nucleic Acid Center, Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Odense M, Denmark.
authors.forename: Jesper
authors.initials: J
authors.lastname: Wengel
authors.affiliation: LEPABE, Laboratory for Process Engineering, Environment, Biotechnology and Energy, Department of Chemical Engineering, Faculty of Engineering, University of Porto, Porto, Portugal. nazevedo@fe.up.pt.
authors.forename: Nuno Filipe
authors.initials: NF
authors.lastname: Azevedo
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D016480:Helicobacter pylori / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D017404:In Situ Hybridization, Fluorescence / Q000295:instrumentation / Q000379:methods*; D015345:Oligonucleotide Probes / Q000235:genetics / Q000378:metabolism; D009841:Oligonucleotides / Q000235:genetics* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D015345:Oligonucleotide Probes; D009841:Oligonucleotides; C477371:locked nucleic acid
keywords: Bacteria; FISH; Locked nucleic acids; Urea