A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

Abstract

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health and performance. Nucleic acid aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward small molecule targets requires special design considerations. This work describes modification and critical steps of a method designed to select structure-switching aptamers to small molecule targets. Binding sequences from a DNA library hybridized to complementary DNA capture probes on magnetic beads are separated from nonbinders via a target-induced change in conformation. This method is advantageous because sequences binding the support matrix (beads) will not be further amplified, and it does not require immobilization of the target molecule. However, the melting temperature of the capture probe and library is kept at or slightly above RT, such that sequences that dehybridize based on thermodynamics will also be present in the supernatant solution. This effectively limits the partitioning efficiency (ability to separate target binding sequences from nonbinders), and therefore many selection rounds will be required to remove background sequences. The reported method differs from previous structure-switching aptamer selections due to implementation of negative selection steps, simplified enrichment monitoring, and extension of the length of the capture probe following selection enrichment to provide enhanced stringency. The selected structure-switching aptamers are advantageous in a gold nanoparticle assay platform that reports the presence of a target molecule by the conformational change of the aptamer. The gold nanoparticle assay was applied because it provides a simple, rapid colorimetric readout that is beneficial in a clinical or deployed environment. Design and optimization considerations are presented for the assay as proof-of-principle work in buffer to provide a foundation for further extension of the work toward small molecule biosensing in physiological fluids.

In Vitro Selection and Characterization of a Single-Stranded DNA Aptamer Against the Herbicide Atrazine

“…The aim of this study was to adopt a previously described Capture-SELEX protocol in order to identify a ssDNA aptamer specific for atrazine that would have a target-induced conformational change. 15 , 16 The ssDNA oligonucleotide library utilized in this study has been successfully screened for multiple aptamers bound to small molecules and protein targets. 7 , 17 − 22 …”

The Use of Aptamers and Molecularly Imprinted Polymers in Biosensors for Environmental Monitoring: A Tale of Two Receptors

“…This is due to the lack of mass-transfer (lack of size difference between bound and unbound DNA) upon binding of the small molecule. Although it is worth noting that structure switching protocols (Reverse SELEX) have overcome this problem through the docking of the DNA library onto a solid phase [27]. Upon binding of the small molecule target, to any sequences within a library, the aptamer undergoes a change in conformation and elutes off the solid phase.…”

Key Aspects of Nucleic Acid Library Design for in Vitro Selection

“…The design of a docking sequence for capture SELEX, namely the length and nucleotide composition, should provide both strong immobilization before target binding and sufficient dissociation afterwards [ 57 ]. As a rule, it is a heterosequence of 12–18 deoxynucleotides (see, e.g., Table 1 ) placed within the random region (as in [ 57 , 78 , 79 ]), or extending one of the primer-binding sites (as described in [ 80 , 81 , 82 , 83 ]). Currently, the capture SELEX strategy is generally employed for DNA selection, but also suits RNA libraries.…”