Molecular and functional characterization of ssDNA aptamers that specifically bind leishmania infantum PABP

Summary A poly (A)-binding protein from Leishmania infantum (LiPABP) has been recently cloned and characterized in our laboratory. Although this protein shows a very high homology with PABPs from other eukaryotic organisms including mammals and other parasites, exist divergences along the sequence t...

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Autores: Guerra-Pérez, Natalia, Ramos, Edurne, García-Hernández, Marta, Pinto, Celia, Soto Álvarez, Manuel, Martín, M. Elena, González, Víctor M.
Tipo de recurso: artículo
Fecha de publicación:2015
País:España
Institución:Universidad Autónoma de Madrid
Repositorio:Biblos-e Archivo. Repositorio Institucional de la UAM
Idioma:inglés
OAI Identifier:oai:repositorio.uam.es:10486/676740
Acceso en línea:http://hdl.handle.net/10486/676740
https://dx.doi.org/10.1371/journal.pone.0140048
Access Level:acceso abierto
Palabra clave:Aptamer
Histone H2A
Polyadenylic acid
Protein binding
Biología y Biomedicina / Biología
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spelling Molecular and functional characterization of ssDNA aptamers that specifically bind leishmania infantum PABPGuerra-Pérez, NataliaRamos, EdurneGarcía-Hernández, MartaPinto, CeliaSoto Álvarez, ManuelMartín, M. ElenaGonzález, Víctor M.AptamerHistone H2APolyadenylic acidProtein bindingBiología y Biomedicina / BiologíaSummary A poly (A)-binding protein from Leishmania infantum (LiPABP) has been recently cloned and characterized in our laboratory. Although this protein shows a very high homology with PABPs from other eukaryotic organisms including mammals and other parasites, exist divergences along the sequence that convert them in potential diagnostic markers and/or therapeutics targets. Aptamers are oligonucleotide ligands that are selected in vitro by their affinity and specificity for the target as a consequence of the particular tertiary structure that they are able to acquire depending on their sequence. Development of high-affinity molecules with the ability to recognize specifically Leishmania proteins is essential for the progress of this kind of study. Results We have selected a ssDNA aptamer population against a recombinant 6xHIS-LiPABP protein (rLiPABP) that is able to recognize the target with a low Kd. Cloning, sequencing and in silico analysis of the aptamers obtained from the population yielded three aptamers (ApPABP#3, ApPABP#7 and ApPABP#11) that significantly bound to PABP with higher affinity than the naïve population. These aptamers were analyzed by ELONA and slot blot to establish affinity and specificity for rLiPABP. Results demonstrated that the three aptamers have high affinity and specificity for the target and that they are able to detect an endogenous LiPABP (eLiPABP) protein amount corresponding to 2500 L. infantum promastigotes in a significant manner. The functional analysis of the aptamers also revealed that ApPABP#11 disrupts the binding of both Myc-LiPABP and eLiPABP to poly (A) in vitro. On the other hand, these aptamers are able to bind and purify LiPABP from complex mixes. Conclusion Results presented here demonstrate that aptamers represent new reagents for characterization of LiPABP and that they can affect LiPABP activity. At this respect, the use of these aptamers as therapeutic tool affecting the physiological role of PABP has to be analyzedThis work was supported by Grants PI05/ 0453 and SAF2010-21663 from the Ministerio de Economia y Competitividad (Spain). MEM and VMG are researchers from FIBio-HRC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Marta Garcia-Hernández is employed by Aptus Biotech SL. The funder provided support in the form of salaries for MG-H, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ sectionPublic Library of ScienceDepartamento de Biología MolecularFacultad de Ciencias20152015-10-12research articlehttp://purl.org/coar/resource_type/c_2df8fbb1VoRhttp://purl.org/coar/version/c_970fb48d4fbd8a85info:eu-repo/semantics/articleapplication/pdfhttp://hdl.handle.net/10486/676740https://dx.doi.org/10.1371/journal.pone.0140048reponame:Biblos-e Archivo. Repositorio Institucional de la UAMinstname:Universidad Autónoma de MadridInglésengopen accesshttp://purl.org/coar/access_right/c_abf2info:eu-repo/semantics/openAccessoai:repositorio.uam.es:10486/6767402026-06-23T12:46:27Z
dc.title.none.fl_str_mv Molecular and functional characterization of ssDNA aptamers that specifically bind leishmania infantum PABP
title Molecular and functional characterization of ssDNA aptamers that specifically bind leishmania infantum PABP
spellingShingle Molecular and functional characterization of ssDNA aptamers that specifically bind leishmania infantum PABP
Guerra-Pérez, Natalia
Aptamer
Histone H2A
Polyadenylic acid
Protein binding
Biología y Biomedicina / Biología
title_short Molecular and functional characterization of ssDNA aptamers that specifically bind leishmania infantum PABP
title_full Molecular and functional characterization of ssDNA aptamers that specifically bind leishmania infantum PABP
title_fullStr Molecular and functional characterization of ssDNA aptamers that specifically bind leishmania infantum PABP
title_full_unstemmed Molecular and functional characterization of ssDNA aptamers that specifically bind leishmania infantum PABP
title_sort Molecular and functional characterization of ssDNA aptamers that specifically bind leishmania infantum PABP
dc.creator.none.fl_str_mv Guerra-Pérez, Natalia
Ramos, Edurne
García-Hernández, Marta
Pinto, Celia
Soto Álvarez, Manuel
Martín, M. Elena
González, Víctor M.
author Guerra-Pérez, Natalia
author_facet Guerra-Pérez, Natalia
Ramos, Edurne
García-Hernández, Marta
Pinto, Celia
Soto Álvarez, Manuel
Martín, M. Elena
González, Víctor M.
author_role author
author2 Ramos, Edurne
García-Hernández, Marta
Pinto, Celia
Soto Álvarez, Manuel
Martín, M. Elena
González, Víctor M.
author2_role author
author
author
author
author
author
dc.contributor.none.fl_str_mv Departamento de Biología Molecular
Facultad de Ciencias
dc.subject.none.fl_str_mv Aptamer
Histone H2A
Polyadenylic acid
Protein binding
Biología y Biomedicina / Biología
topic Aptamer
Histone H2A
Polyadenylic acid
Protein binding
Biología y Biomedicina / Biología
description Summary A poly (A)-binding protein from Leishmania infantum (LiPABP) has been recently cloned and characterized in our laboratory. Although this protein shows a very high homology with PABPs from other eukaryotic organisms including mammals and other parasites, exist divergences along the sequence that convert them in potential diagnostic markers and/or therapeutics targets. Aptamers are oligonucleotide ligands that are selected in vitro by their affinity and specificity for the target as a consequence of the particular tertiary structure that they are able to acquire depending on their sequence. Development of high-affinity molecules with the ability to recognize specifically Leishmania proteins is essential for the progress of this kind of study. Results We have selected a ssDNA aptamer population against a recombinant 6xHIS-LiPABP protein (rLiPABP) that is able to recognize the target with a low Kd. Cloning, sequencing and in silico analysis of the aptamers obtained from the population yielded three aptamers (ApPABP#3, ApPABP#7 and ApPABP#11) that significantly bound to PABP with higher affinity than the naïve population. These aptamers were analyzed by ELONA and slot blot to establish affinity and specificity for rLiPABP. Results demonstrated that the three aptamers have high affinity and specificity for the target and that they are able to detect an endogenous LiPABP (eLiPABP) protein amount corresponding to 2500 L. infantum promastigotes in a significant manner. The functional analysis of the aptamers also revealed that ApPABP#11 disrupts the binding of both Myc-LiPABP and eLiPABP to poly (A) in vitro. On the other hand, these aptamers are able to bind and purify LiPABP from complex mixes. Conclusion Results presented here demonstrate that aptamers represent new reagents for characterization of LiPABP and that they can affect LiPABP activity. At this respect, the use of these aptamers as therapeutic tool affecting the physiological role of PABP has to be analyzed
publishDate 2015
dc.date.none.fl_str_mv 2015
2015-10-12
dc.type.none.fl_str_mv research article
http://purl.org/coar/resource_type/c_2df8fbb1
VoR
http://purl.org/coar/version/c_970fb48d4fbd8a85
dc.type.openaire.fl_str_mv info:eu-repo/semantics/article
format article
dc.identifier.none.fl_str_mv http://hdl.handle.net/10486/676740
https://dx.doi.org/10.1371/journal.pone.0140048
url http://hdl.handle.net/10486/676740
https://dx.doi.org/10.1371/journal.pone.0140048
dc.language.none.fl_str_mv Inglés
eng
language_invalid_str_mv Inglés
language eng
dc.rights.none.fl_str_mv open access
http://purl.org/coar/access_right/c_abf2
dc.rights.openaire.fl_str_mv info:eu-repo/semantics/openAccess
rights_invalid_str_mv open access
http://purl.org/coar/access_right/c_abf2
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv Public Library of Science
publisher.none.fl_str_mv Public Library of Science
dc.source.none.fl_str_mv reponame:Biblos-e Archivo. Repositorio Institucional de la UAM
instname:Universidad Autónoma de Madrid
instname_str Universidad Autónoma de Madrid
reponame_str Biblos-e Archivo. Repositorio Institucional de la UAM
collection Biblos-e Archivo. Repositorio Institucional de la UAM
repository.name.fl_str_mv
repository.mail.fl_str_mv
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