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

Barfod, Anders LU ; Singh, Birendra LU ; Johanson, Urban LU ; Riesbeck, Kristian LU and Kjellbom, Per LU (2014) In Molecular Biotechnology 56(8). p.714-725
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... (More)
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. (Less)
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author
organization
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type
Contribution to journal
publication status
published
subject
keywords
Adhesins, Bacterial, Aptamers, Nucleotide, Base Sequence, Biotechnology, DNA, Bacterial, Haemophilus influenzae, Humans, In Vitro Techniques, Molecular Sequence Data, Nucleic Acid Conformation, Protein Binding, SELEX Aptamer Technique, Surface Plasmon Resonance, Vitronectin, Journal Article, Research Support, Non-U.S. Gov't
in
Molecular Biotechnology
volume
56
issue
8
pages
12 pages
publisher
Humana Press
external identifiers
  • pmid:24682699
  • wos:000339869800004
  • scopus:84904855863
ISSN
1559-0305
DOI
10.1007/s12033-014-9749-xhttp://dx.doi.org/10.1007/s12033-014-9749-x
language
English
LU publication?
yes
id
8c7e40d1-16a3-4736-b012-fa9ac5daaac6 (old id 4431486)
alternative location
http://www.ncbi.nlm.nih.gov/pubmed/24682699?dopt=Abstract
date added to LUP
2014-05-07 19:18:53
date last changed
2017-01-01 03:38:12
@article{8c7e40d1-16a3-4736-b012-fa9ac5daaac6,
  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.},
  author       = {Barfod, Anders and Singh, Birendra and Johanson, Urban and Riesbeck, Kristian and Kjellbom, Per},
  issn         = {1559-0305},
  keyword      = {Adhesins, Bacterial,Aptamers, Nucleotide,Base Sequence,Biotechnology,DNA, Bacterial,Haemophilus influenzae,Humans,In Vitro Techniques,Molecular Sequence Data,Nucleic Acid Conformation,Protein Binding,SELEX Aptamer Technique,Surface Plasmon Resonance,Vitronectin,Journal Article,Research Support, Non-U.S. Gov't},
  language     = {eng},
  number       = {8},
  pages        = {714--725},
  publisher    = {Humana Press},
  series       = {Molecular Biotechnology},
  title        = {In Vitro Selection of RNA Aptamers Directed Against Protein E: A Haemophilus influenzae Adhesin. : a Haemophilus influenzae adhesin},
  url          = {http://dx.doi.org/10.1007/s12033-014-9749-xhttp://dx.doi.org/10.1007/s12033-014-9749-x},
  volume       = {56},
  year         = {2014},
}