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Guerra-Perez N.,Laboratory of Aptamers | Guerra-Perez N.,Center for Biomedical Research | Ramos E.,Laboratory of Aptamers | Garcia-Hernandez M.,Aptus Biotech SL | And 4 more authors.
PLoS ONE | Year: 2015

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. © 2015 Guerra-Pérez et al.This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Source

Rodriguez P.,CSIC - National Center for Biotechnology | Rodriguez P.,CIBER ISCIII | Perez-Morgado M.I.,Laboratory of Aptamers | Gonzalez V.M.,Laboratory of Aptamers | And 3 more authors.
Molecular Therapy - Nucleic Acids | Year: 2016

The genetic diversity of the influenza virus hinders the use of broad spectrum antiviral drugs and favors the appearance of resistant strains. Single-stranded DNA aptamers represent an innovative approach with potential application as antiviral compounds. The mRNAs of influenza virus possess a 5′cap structure and a 3′poly(A) tail that makes them structurally indistinguishable from cellular mRNAs. However, selective translation of viral mRNAs occurs in infected cells through a discriminatory mechanism, whereby viral polymerase and NS1 interact with components of the translation initiation complex, such as the eIF4GI and PABP1 proteins. We have studied the potential of two specific aptamers that recognize PABP1 (ApPABP7 and ApPABP11) to act as anti-influenza drugs. Both aptamers reduce viral genome expression and the production of infective influenza virus particles. The interaction of viral polymerase with the eIF4GI translation initiation factor is hindered by transfection of infected cells with both PABP1 aptamers, and ApPABP11 also inhibits the association of NS1 with PABP1 and eIF4GI. These results indicate that aptamers targeting the host factors that interact with viral proteins may potentially have a broad therapeutic spectrum, reducing the appearance of escape mutants and resistant subtypes. Source

Garcia-Recio E.M.,Laboratory of Aptamers | Pinto-Diez C.,Laboratory of Aptamers | Perez-Morgado M.I.,Laboratory of Aptamers | Garcia-Hernandez M.,Aptus Biotech SL | And 3 more authors.
Molecular Therapy - Nucleic Acids | Year: 2016

Elevated expression levels of eukaryotic initiation factor 4E (eIF4E) promote cancer development and progression. MAP kinase interacting kinases (MNKs) modulate the function of eIF4E through the phosphorylation that is necessary for oncogenic transformation. Therefore, pharmacologic MNK inhibitors may provide a nontoxic and effective anticancer strategy. MNK1b is a truncated isoform of MNK1a that is active in the absence of stimuli. Using in vitro selection, high-affinity DNA aptamers to MNK1b were selected from a library of ssDNA. Selection was monitored using the enzyme-linked oligonucleotide assay (ELONA), and the selected aptamer population was cloned and sequenced. Four groups of aptamers were identified, and the affinities of one representative for rMNK1b were determined using ELONA and quantitative polymerase chain reaction. Two aptamers, named apMNK2F and apMNK3R, had a lower Kd in the nmol/l range. The secondary structure of the selected aptamers was predicted using mFold, and the QGRS Mapper indicated the presence of potential G-quadruplex structures in both aptamers. The selected aptamers were highly specific against MNK1, showing higher affinity to MNK1b than to MNK1a. Interestingly, both aptamers were able to produce significant translation inhibition and prevent tumor cell proliferation and migration and colony formation in breast cancer cells. These results indicate that MNK1 aptamers have an attractive therapeutic potential. Source

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