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

• The strategy carried out a visual assay for NAD+.
• A strategy based on ligase-mediated inhibition on strand displacement amplification.
• The colorimetric assay is simple, high sensitivity and high selectivity.
• A novel platform for investigating cofactors, small molecules and DNA ligases.

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.

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