In vitro isolation of class-specific oligonucleotide-based small-molecule receptors

Abstract

Class-specific bioreceptors are highly desirable for recognizing structurally similar small molecules, but the generation of such affinity elements has proven challenging. We here develop a novel ‘parallel-and-serial’ selection strategy for isolating class-specific oligonucleotide-based receptors (aptamers) in vitro. This strategy first entails parallel selection to selectively enrich cross-reactive binding sequences, followed by serial selection that enriches aptamers binding to a designated target family. As a demonstration, we isolate a class-specific DNA aptamer against a family of designer drugs known as synthetic cathinones. The aptamer binds to 12 diverse synthetic cathinones with nanomolar affinity and does not respond to 11 structurally similar non-target compounds, some of which differ from the cathinone targets by a single atom. This is the first account of an aptamer exhibiting a combination of broad target cross-reactivity, high affinity and remarkable specificity. Leveraging the qualities of this aptamer, instantaneous colorimetric detection of synthetic cathinones at nanomolar concentrations in biological samples is achieved. Our findings significantly expand the binding capabilities of aptamers as class-specific bioreceptors and further demonstrate the power of rationally designed selection strategies for isolating customized aptamers with desired binding profiles. We believe that our aptamer isolation approach can be broadly applied to isolate class-specific aptamers for various small molecule families.

Antibodies, Nanobodies, or Aptamers—Which Is Best for Deciphering the Proteomes of Non-Model Species?

“…This process was coined "toggle SELEX" and involved interchanging the protein in selection rounds between the two. There have since been several adaptations to this protocol to generate cross-species binding aptamers [75][76][77].…”

in vitro Selection of Aptamer for Imidacloprid Recognition as Model Analyte and Construction of a Water Analysis Platform

“…Moreover, the aptasensor platform could be useful for identification not only Imi, but also its metabolite 6-CN. For future studies, Apta-1 is promising for engineering of class-selective aptamers [67] that may be useful for detection of Imi metabolites [68].…”

Label-free profiling of DNA aptamer-small molecule binding using T5 exonuclease

“…For example, accurate diagnostic detection of disease-related analytes or biomarkers in biological specimens requires aptamers that bind strongly to a single target without any cross-reactivity to the myriad of interferents commonly present in complex biological matrices. On the other hand, applications that require the detection of large families of structurally-related compounds such as antibiotics ( 10 ) or illicit drugs ( 11 ) require aptamers with high affinity and broad cross-reactivity to the target family, but with tightly controlled specificity against those outside that family of compounds. However, finding aptamers with satisfactory binding profiles for these various applications is a challenging task.…”

“…However, finding aptamers with satisfactory binding profiles for these various applications is a challenging task. After several rounds of SELEX, tens to hundreds of aptamer candidates ( 11–13 ) can be identified through DNA sequencing methods such as Sanger sequencing or high-throughput sequencing on the basis of their prevalence or degree of enrichment ( 14 ). However, the binding properties of these candidates is not readily apparent, and a thorough comprehensive characterization of the affinity of each candidate sequence towards the target(s) and relevant interferents is the only means of identifying suitable aptamers.…”

“…Alternatively, dye-displacement assays offer a label-free approach. Certain small-molecule-binding aptamers have the capability of binding dyes such as thiazole orange ( 20 , 21 ), SYBR Green I ( 22 ), and diethylthiacarbocyanine (also known as Cy7) ( 11 , 23 , 24 ). In some cases, the binding of a ligand to aptamer-dye complexes can induce displacement of the dye, which results in a concomitant change in the fluorescence or absorbance of the dye that can in turn be used to assess aptamer binding.…”