One-step label-free chemiluminescent assay for determination of exonuclease III activity towards hairpin oligonucleotides

Enzyme Microb Technol. 2019 Dec:131:109419. doi: 10.1016/j.enzmictec.2019.109419. Epub 2019 Aug 30.

Abstract

Fast label-free chemiluminescent assay for determination of exonuclease III (ExoIII) activity measured towards hairpin oligonucleotide substrates was developed. The designed substrates consisted of EAD2 aptamer to hemin which was associated with DNA sequence complementary to 5'-terminus fragment of EAD2. In the presence of ExoIII the associated sequence of the hairpin stem was digested, producing EAD2 aptamer which reacted with hemin with the formation of peroxidase-mimicking DNAzyme (PMDNAzyme). The catalytic activity of the produced PMDNAzyme was measured towards luminol/H2O2. Under the optimized conditions the limit of detection and sensitivity of the one-step chemiluminescent assay of ExoIII were 7.3 nM and 1.7 × 108 M-1, respectively. The coefficient of variation (CV) was lower than 6%.

Keywords: Chemiluminescence; DNAzyme; Exonuclease III; Label-free assay; Peroxidase mimetics; Peroxidase-mimicking.

MeSH terms

  • Aptamers, Nucleotide / metabolism
  • Exodeoxyribonucleases / analysis*
  • Hemin / metabolism
  • Luminescent Measurements / methods*
  • Nucleic Acid Conformation
  • Oligonucleotides / chemistry
  • Oligonucleotides / metabolism*
  • Sensitivity and Specificity

Substances

  • Aptamers, Nucleotide
  • Oligonucleotides
  • Hemin
  • Exodeoxyribonucleases
  • exodeoxyribonuclease III