Label-free detection of nicotinamide adenine dinucleotide based on ligation-triggered exonuclease III-assisted signal amplification

Abstract

Effective detection of nicotinamide adenine dinucleotide (NAD⁺) is crucial for better understanding its roles in biological process and further validating its function in clinical diagnosis. Herein, we developed a ligation-triggered exonuclease III (Exo III)-assisted signal amplification strategy for label-free and sensitive detection of NAD⁺. We ingeniously designed an oligo1–oligo2–cDNA double-strand DNA (dsDNA) probe and a G-quadruplex (G4)-template dsDNA probe. In the presence of NAD⁺, oligo1–oligo2–cDNA dsDNA probe containing a ligatable nick was ligated by E. coli DNA ligase and formed a complete trigger DNA (tDNA). Then, Exo III digested the ligated oligo1–oligo2–cDNA dsDNA probe and released the formed tDNA. Successively, tDNA hybridized with the G4-template dsDNA probe and initiated the Exo III-assisted cycling cleavage, ultimately releasing tDNA and G4 DNA. Finally, the released large amounts of G4 DNA interacted with N-methylmesoporphyrin IX (NMM) to form G4–NMM complex, generating a label-free and enhanced fluorescence signal. The proposed assay provided a detection limit as low as 3.0 pM and exhibited high selectivity against NAD⁺ analogues. Thus, the strategy provided a facile and convenient tool for sensitive quantification of NAD⁺ in NAD⁺ related biological processes and clinical diagnosis.