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

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.

Aptamers, the Nucleic Acid Antibodies, in Cancer Therapy

“…Aptamers can form diverse three-dimensional structures and can thus bind to a variety of targets specifically and tightly. The binding affinities (dissociation constants) of aptamers are usually in the range of low nanomolar to high picomolar but may reach femtomolar [7,8]. The recognition is highly exquisite and can discriminate structural differences between enantiomers (mirror images with identical chemical composition) and the presence or absence of a hydroxyl group [9].…”

The Complex Molecules Detector (CMOLD): A Fluidic-Based Instrument Suite to Search for (Bio)chemical Complexity on Mars and Icy Moons

“…Direct and competitive aptamer-based assay for detecting low MW compounds and small epitope-containing polymers. We have developed Aps to specifically bind different kind of biomolecules, including structured nucleic acids (Sanchez-Luque et al, 2014), proteins (Moreno et al, 2019), and low MW compounds such as amino acids and antibiotics. We have also used some of our protein-specific ssDNA aptamers as bioaffinity probes to develop graphenebased aptasensors (Bueno et al, 2019).…”

“…Similarly, we have developed specific aptamers to bind structured nucleic acids (Sanchez-Luque et al, 2014), proteins (Moreno et al, 2019), as well as some amino acids, antibiotics, and peptides (not shown). We have also tested the performance of aptamers as capture probes in different kinds of biosensors (Bueno et al, 2019).…”

A Bottom-Up Approach for Developing Aptasensors for Abused Drugs: Biosensors in Forensics

“…Binding affinity of aptamers is expressed as dissociation constant (K d ) that can be determined by various techniques, such as surface plasmon resonance (SPR) [59], isothermal titration calorimetry (ITC) [60], fluorescence spectroscopy [61,62], equilibrium dialysis [63], backscattering interferometry (BSI) [64], enzyme-linked oligonucleotide assay (ELONA) [65], and combined ELONA real-time quantitative PCR (qPCR) [66]. Post-SELEX optimization via truncation, chemical modification of nucleobases, or mutagenesis can be performed to improve the binding affinity or sensor properties [67].…”