Aptamer-based detection of adenosine triphosphate via qPCR

Talanta. 2017 Sep 1:172:199-205. doi: 10.1016/j.talanta.2017.05.037. Epub 2017 May 18.

Abstract

Sensitive and specific detection and quantification of small molecules often remain challenging. We developed a novel magnetic bead-based aptamer-assisted real-time PCR (Apta-qPCR) assay to provide a versatile platform for quantification of small molecules. The assay has been realized for the detection of ATP as a model system. The assay relies on a combination of qPCR with the target-induced dissociation (TID) of ATP aptamer from an oligonucleotide, complementary to the ATP binding site of the aptamer. The complementary oligonucleotide was immobilized on deoxythymidine (dT)-modified magnetic beads (dT-beads) and hybridized with the aptamer. The presence of ATP resulted in dissociation of the aptamer from the dT-beads and the dissociated aptamer was quantified using qPCR. The Apta-qPCR assay was able to detect 17nM ATP with a broad dynamic range from 50nM to 5mM. The assay is label-free, and real-time PCR-based detection of aptamer facilitates high sensitivity. The presented method is highly versatile and can be applied to various aptamer-target pairs to allow detection of a broad range of target analytes.

Keywords: ATP; Apta-qPCR; Aptamer; Small molecules; Target-induced dissociation.

MeSH terms

  • Adenosine Triphosphate / analysis*
  • Adenosine Triphosphate / metabolism
  • Aptamers, Nucleotide / metabolism*
  • HeLa Cells
  • Humans
  • Limit of Detection
  • Real-Time Polymerase Chain Reaction / methods*

Substances

  • Aptamers, Nucleotide
  • Adenosine Triphosphate