A simple and highly sensitive DNAzyme-based assay for nicotinamide adenine dinucleotide by ligase-mediated inhibition of strand displacement amplification.

Existing strategies for detecting nicotinamide adenine dinucleotide (NAD(+)) or other cofactors are commonly cumbersome and moderate sensitive. We report a novel DNAzyme-based visual assay strategy for NAD(+) based on ligase-mediated inhibition of the strand displacement amplification (SDA). In the presence of NAD(+), the SDA can be inhibited by the ligase reaction of two primers, which can initiate the SDA reaction in the case of no ligation, resulting in a dramatically decreasing yield of the SDA product, a G-quadruplex DNAzyme that can quantitatively catalyze the formation of a colored product. Therefore, the quantitative analysis for NAD(+) can be achieved visually with high sensitivity. The developed strategy provides a simple colorimetric approach with high selectivity against most interferences and a detection limit as low as 50 pM. It also provides a universal platform for investigating cofactors or other related small molecules as well as quantifying the activity of DNA ligases.