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In Vitro Selection of RNA Aptamers Directed Against Protein E: A Haemophilus influenzae Adhesin

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Abstract

Protein E (PE) of Haemophilus influenzae is a highly conserved ubiquitous surface protein involved in adhesion to and activation of epithelial cells. The host proteins—vitronectin, laminin, and plasminogen are major targets for PE-dependent interactions with the host. To identify novel inhibitory molecules of PE, we used an in vitro selection method based on systematic evolution of ligands by exponential enrichment known as SELEX in order to select 2′F-modified RNA aptamers that specifically bind to PE. Fourteen selection cycles were performed with decreasing concentrations of PE. Sequencing of clones from the 14th selection round revealed the presence of semiconserved sequence motifs in loop regions of the RNA aptamers. Among these, three aptamers showed the highest affinity to PE in electrophoretic mobility shift assays and in dot blots. These three aptamers also inhibited the interaction of PE with vitronectin as revealed by ELISA. Moreover, pre-treatment of H. influenzae with the aptamers significantly inhibited binding of vitronectin to the bacterial surface. Biacore experiments indicated that one of the aptamers had a higher binding affinity for PE as compared to the other aptamers. Our results show that it is possible to select RNA inhibitors against bacterial adhesins using SELEX in order to inhibit interactions with target proteins.

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Acknowledgments

This work was supported by the Swedish Research Council (VR; grant number 521-2010-4221, www.vr.se), the Research Council Formas, the Research School in Pharmaceutical Sciences (FLÄK), the Swedish Medical Research Council, the Alfred Österlund, the Anna and Edwin Berger, Anna-Lisa and Sven-Erik Lundgren, Greta and Johan Kock, the Physiographical Society, the Cancer Foundation at the University Hospital in Malmö, and Skåne County Council′s research and development foundation.

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Authors and Affiliations

  1. Department of Biochemistry and Structural Biology, CMPS, Lund University, Box 124, 221 00, Lund, Sweden

    Anders Barfod, Urban Johanson & Per Kjellbom

  2. Medical Microbiology, Department of Laboratory Medicine Malmö, Lund University, Skåne University Hospital, 205 02, Malmö, Sweden

    Birendra Singh & Kristian Riesbeck

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  1. Anders Barfod
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  2. Birendra Singh
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  3. Urban Johanson
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  4. Kristian Riesbeck
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Correspondence to Anders Barfod.

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Barfod, A., Singh, B., Johanson, U. et al. In Vitro Selection of RNA Aptamers Directed Against Protein E: A Haemophilus influenzae Adhesin. Mol Biotechnol 56, 714–725 (2014). https://doi.org/10.1007/s12033-014-9749-x

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