The Arsenic-Binding Aptamer Cannot Bind Arsenic: Critical Evaluation of Aptamer Selection and Binding

Abstract

An arsenic-binding aptamer named Ars-3 was reported in 2009, and it has been used for detection of As(III) in more than two dozen papers. In this work, we performed extensive binding assays using isothermal titration calorimetry, various DNA-staining dyes, and gold nanoparticles. By carefully comparing Ars-3 and a few random control DNA sequences, no specific binding of As(III) was observed in each case. Therefore, we conclude that Ars-3 cannot bind As(III). Possible reasons for some of the previously reported binding and detection were speculated to be related to the adsorption of As(III) onto gold surfaces, which were used in many related sensor designs, and As(III)/Au interactions were not considered before. The selection data in the original paper were then analyzed in terms of sequence alignment, secondary structure prediction, and dissociation constant measurement. These steps need rigorous testing before confirming specific binding of newly selected aptamers. This study calls for attention to the gap between aptamer selection and biosensor design, and the gap needs to be filled by careful binding assays to further the growth of the aptamer field.

Preliminary Data on a SERS-Responsive Sensor Based on Metallic Nanostructures Functionalized by Aptamers Specific for Arsenic

“…Indeed, in parallel to our experiments two very recent works demonstrated the high affinity of arsenite for gold and the false positive generated from the interactions of aptamer and As III on gold surface-involving assays [19][20]. Particularly, in ref [20], the ability of aptamer Ars3 to bind arsenite and arsenate was confuted by accurate ITC (Isothermal Titration Calorimetry) measurements. Thus, our difficulty to reproduce the binding assay of Ars3 with As III on an agarose affinity resin (instead of the original gold-based SPR assay) was actually due to the absence of affinity of the aptamer for the arsenic target, as well as to the partial detachment from the resin of electrostatically-immobilized arsenic after repeated washings of the resin with buffered solutions.…”

“…Thus, on the basis of the recent literature reports [19][20] and of our scientific results, we can definitely exclude the DNA aptamer Ars3 as a candidate for specifically bind arsenic in a SERSbased sensor. Our studies are now directed towards peptide aptamers, specifically focussing on…”

“…Thus, we found many contradictory results with respect to the first work on the arsenic binding aptamer Ars3 [8]. Indeed, in parallel to our experiments two very recent works demonstrated the high affinity of arsenite for gold and the false positive generated from the interactions of aptamer and As III on gold surface-involving assays [19][20]. Particularly, in ref [20], the ability of aptamer Ars3 to bind arsenite and arsenate was confuted by accurate ITC (Isothermal Titration Calorimetry) measurements.…”

Label‐Free Colorimetric Biosensors Based on Aptamers and Gold Nanoparticles: A Critical Review

“…By extensive binding assays, we did not believe this sequence can actually bind As(III). [76] Nevertheless, an As(III)dependent color change was still observed with this sequence for the reasons to be discussed in the next section. There are other examples for the detection of Pb 2 + , [77,78] and UO 2 2 + based on DNAzyme cleavage, [79] which will not be covered in this review.…”

High-efficiency enrichment enables identification of aptamers to circulating Plasmodium falciparum-infected erythrocytes

“…Malaria.1 displayed binding toward IEs in the nanomolar range (apparent K d ~60 nM). Given recent concerns of the functionality of selected aptamers 42 , we confirmed that the apparent binding is a result of a specific aptamer interaction by performing the binding assay using a random DNA oligonucleotide sequence of the same length, RDM1. RDM1 showed minimal binding to IEs up to concentrations of 300 nM (Fig.…”