In vitro isolation of small-molecule-binding aptamers with intrinsic dye-displacement functionality

Nucleic Acids Res. 2018 May 4;46(8):e43. doi: 10.1093/nar/gky026.

Abstract

Aptamer-based sensors offer a powerful tool for molecular detection, but the practical implementation of these biosensors is hindered by costly and laborious sequence engineering and chemical modification procedures. We report a simple strategy for directly isolating signal-reporting aptamers in vitro through systematic evolution of ligands by exponential enrichment (SELEX) that transduce binding events into a detectable change of absorbance via target-induced displacement of a small-molecule dye. We first demonstrate that diethylthiatricarbocyanine (Cy7) can stack into DNA three-way junctions (TWJs) in a sequence-independent fashion, greatly altering the dye's absorbance spectrum. We then design a TWJ-containing structured library and isolate an aptamer against 3,4-methylenedioxypyrovalerone (MDPV), a synthetic cathinone that is an emerging drug of abuse. This aptamer intrinsically binds Cy7 within its TWJ domain, but MDPV efficiently displaces the dye, resulting in a change in absorbance within seconds. This assay is label-free, and detects nanomolar concentrations of MDPV. It also recognizes other synthetic cathinones, offering the potential to detect newly-emerging designer drugs, but does not detect structurally-similar non-cathinone compounds or common cutting agents. Moreover, we demonstrate that the Cy7-displacement colorimetric assay is more sensitive than a conventional strand-displacement fluorescence assay. We believe our strategy offers an effective generalized approach for the development of sensitive dye-displacement colorimetric assays for other small-molecule targets.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Aptamers, Nucleotide / chemistry
  • Aptamers, Nucleotide / genetics
  • Aptamers, Nucleotide / isolation & purification*
  • Base Sequence
  • Benzodioxoles / analysis
  • Biosensing Techniques / methods
  • Carbocyanines / chemistry
  • Colorimetry / methods
  • Coloring Agents / chemistry
  • DNA / chemistry
  • DNA / genetics
  • Molecular Structure
  • Pyrrolidines / analysis
  • SELEX Aptamer Technique / methods*
  • Synthetic Cathinone

Substances

  • Aptamers, Nucleotide
  • Benzodioxoles
  • Carbocyanines
  • Coloring Agents
  • Pyrrolidines
  • indotricarbocyanine
  • DNA
  • Synthetic Cathinone