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.

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Correspondence to Nuno Filipe Azevedo.

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Funding

This study was funded by Project UID/EQU/00,511/2013-LEPABE, by the FCT/MEC with national funds and when applicable co-funded by FEDER in the scope of the P2020 Partnership Agreement Project NORTE-07-0124-FEDER-000,025-RL2_Environment&Health; by FEDER funds through Programa Operacional Factores de Competitividade–COMPETE; by the Programa Operacional do Norte (ON2) program and by national funds through FCT-Fundação para a Ciência e a Tecnologia (DNA mimics Research Project PIC/IC/82,815/2007), PhD grant (SFRH/BD/72,999/2010 to SF), and Post-Doctoral fellowship (SFRH/BPD/78,846/2011 to NG); and by Nucleic Acid Center, University of Southern Denmark.

Conflict of interest

Jesper Wengel is cofounder of RiboTask ApS, which offers LNA/2′-OMe-RNA probes for RNA targeting. Nuno Filipe Azevedo is cofounder of Biomode SA, which develops molecular methods for the rapid detection of microorganisms. This does not alter the authors’ adherence to all the Applied Microbiology and Biotechnology policies on sharing data and materials.

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This article does not contain any studies with human participants or animals performed by any of the authors.

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Fontenete, S., Carvalho, D., Guimarães, N. et al. Application of locked nucleic acid-based probes in fluorescence in situ hybridization. Appl Microbiol Biotechnol 100, 5897–5906 (2016). https://doi.org/10.1007/s00253-016-7429-4

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Keywords

  • FISH
  • Locked nucleic acids
  • Urea
  • Bacteria