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Article

A Combined ELONA-(RT)qPCR Approach for Characterizing DNA and RNA Aptamers Selected against PCBP-2

1
Laboratory of Molecular Evolution, Centro de Astrobiología (CSIC-INTA), Torrejón de Ardoz, 28850 Madrid, Spain
2
Centro de Biología Molecular “Severo Ochoa” (CSIC-UAM), 28049 Madrid, Spain
3
Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), 28029 Madrid, Spain
*
Author to whom correspondence should be addressed.
Molecules 2019, 24(7), 1213; https://doi.org/10.3390/molecules24071213
Received: 27 February 2019 / Revised: 24 March 2019 / Accepted: 26 March 2019 / Published: 28 March 2019
(This article belongs to the Section Analytical Chemistry)
Improvements in Systematic Evolution of Ligands by EXponential enrichment (SELEX) technology and DNA sequencing methods have led to the identification of a large number of active nucleic acid molecules after any aptamer selection experiment. As a result, the search for the fittest aptamers has become a laborious and time-consuming task. Herein, we present an optimized approach for the label-free characterization of DNA and RNA aptamers in parallel. The developed method consists in an Enzyme-Linked OligoNucleotide Assay (ELONA) coupled to either real-time quantitative PCR (qPCR, for DNA aptamers) or reverse transcription qPCR (RTqPCR, for RNA aptamers), which allows the detection of aptamer-target interactions in the high femtomolar range. We have applied this methodology to the affinity analysis of DNA and RNA aptamers selected against the poly(C)-binding protein 2 (PCBP-2). In addition, we have used ELONA-(RT)qPCR to quantify the dissociation constant (Kd) and maximum binding capacity (Bmax) of 16 high affinity DNA and RNA aptamers. The Kd values of the high affinity DNA aptamers were compared to those derived from colorimetric ELONA performed in parallel. Additionally, Electrophoretic Mobility Shift Assays (EMSA) were used to confirm the binding of representative PCBP-2-specific RNA aptamers in solution. We propose this ELONA-(RT)qPCR approach as a general strategy for aptamer characterization, with a broad applicability in biotechnology and biomedicine. View Full-Text
Keywords: aptamer; SELEX; in vitro selection; ELONA; ELASA; qPCR; RTqPCR; PCBP-2 aptamer; SELEX; in vitro selection; ELONA; ELASA; qPCR; RTqPCR; PCBP-2
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MDPI and ACS Style

Moreno, M.; Fernández-Algar, M.; Fernández-Chamorro, J.; Ramajo, J.; Martínez-Salas, E.; Briones, C. A Combined ELONA-(RT)qPCR Approach for Characterizing DNA and RNA Aptamers Selected against PCBP-2. Molecules 2019, 24, 1213. https://doi.org/10.3390/molecules24071213

AMA Style

Moreno M, Fernández-Algar M, Fernández-Chamorro J, Ramajo J, Martínez-Salas E, Briones C. A Combined ELONA-(RT)qPCR Approach for Characterizing DNA and RNA Aptamers Selected against PCBP-2. Molecules. 2019; 24(7):1213. https://doi.org/10.3390/molecules24071213

Chicago/Turabian Style

Moreno, Miguel, María Fernández-Algar, Javier Fernández-Chamorro, Jorge Ramajo, Encarnación Martínez-Salas, and Carlos Briones. 2019. "A Combined ELONA-(RT)qPCR Approach for Characterizing DNA and RNA Aptamers Selected against PCBP-2" Molecules 24, no. 7: 1213. https://doi.org/10.3390/molecules24071213

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