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)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/4431486
- author
- Barfod, Anders
LU
; Singh, Birendra
LU
; Johanson, Urban
LU
; Riesbeck, Kristian
LU
and Kjellbom, Per
LU
- organization
- publishing date
- 2014-08
- 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
- pmid:24682699
- ISSN
- 1559-0305
- DOI
- 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
- 2016-04-01 10:29:09
- date last changed
- 2022-01-25 23:43:02
@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}},
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}},
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-x}},
doi = {{10.1007/s12033-014-9749-x}},
volume = {{56}},
year = {{2014}},
}