Molecular and functional characterization of ssDNA aptamers that specifically bind leishmania infantum PABP
Entity
UAM. Departamento de Biología MolecularPublisher
Public Library of ScienceDate
2015-10-12Citation
10.1371/journal.pone.0140048
PLoS ONE 10.10 (2015): e0140048
ISSN
1932-6203 (print)DOI
10.1371/journal.pone.0140048Funded by
This 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’ sectionProject
Gobierno de España. SAF2010-21663; Gobierno de España. Grants PI05/0453Editor's Version
http://dx.doi.org/10.1371/journal.pone.0140048Subjects
Aptamer; Histone H2A; Polyadenylic acid; Protein binding; Biología y Biomedicina / BiologíaRights
© 2015 Guerra-Pérez et alAbstract
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
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Google Scholar:Guerra-Pérez, Natalia
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Ramos, Edurne
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García-Hernández, Marta
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Pinto, Celia
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Soto Álvarez, Manuel
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Martín, M. Elena
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González, Víctor M.
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