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

Miguel Moreno,Carlos Briones,Encarnación Martínez-Salas,Javier Fernández-Chamorro,María Fernández-Algar,Jorge Ramajo

doi:10.3390/molecules24071213

Abstract

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.

Highlights

Aptamers are single-stranded DNA or RNA molecules that can bind with high affinity and specificity to a desired target molecule [1]
Target poly(C)-binding protein 2 (PCBP-2) was expressed as a fusion protein with a polyhistidine (6xHis) tag at its N-terminal end, allowing itswas immobilization onto
PCBP-2 expressed as a fusion protein with a polyhistidine

Summary

Introduction

Aptamers are single-stranded (ss) DNA or RNA molecules that can bind with high affinity and specificity to a desired target molecule [1]. They are isolated from a large library of synthetic random oligonucleotides using an amplification-selection in vitro process termed Systematic Evolution of Ligands by EXponential enrichment (SELEX) [2]. DNA and RNA aptamers are able to recognize and, eventually, alter the activity of their target molecules by establishing non-covalent aptamer–target molecular interactions. The current SELEX technology allows the generation of aptamers with high affinity and specificity for a broad range of targets, including low molecular weight compounds, peptides, proteins, structured nucleic acids, Molecules 2019, 24, 1213; doi:10.3390/molecules24071213 www.mdpi.com/journal/molecules. Aptamers have being used in a growing number of biotechnological and biomedical applications over the last decade [4]

Methods

Results

Discussion

Conclusion