Binding mediated MNAzyme signal amplification strategy for enzyme-free and label-free detection of DNA-binding proteins

Highlights: • An enzyme-free and label-free method was proposed for DNA-binding protein detection. • The assay employed binding mediated MNAzyme signal amplification strategy. • G-quadruplex based light-up fluorescence switch was used for signal amplification. • The assay is suitable for accurate detection of NF-κB p50 in human serum samples. A novel MNAzyme signal amplification strategy was developed for enzyme-free and label-free detection of DNA-binding proteins. This strategy relied on the binding-mediated MNAzyme cleavage and G-quadruplex-based light-up fluorescence switch. Three DNA sequences were designed to construct the MNAzyme in which DNA1 (including half binding site of the target protein and a toehold sequence) and DNA2 (including another half binding site of the target protein and one MNAzyme partzyme) firstly hybridized. The target protein recognized the binding sites on DNA1-DNA2 hybrid to form a stable protein-DNA1-DNA2 conjugates. Then, the MNAzyme was assembled with the presence of DNA3 which contained another MNAzyme partzyme and the complementary sequence of DNA1. The active MNAzyme cleaved DNA4 to release the G-quadruplex that was locked in the stem of DNA4. Finally, N-methyl mesoporphyrin IX (NMM) was inserted into the released G-quadruplex structure and the fluorescence signal was turned on. Taking nuclear factor-κB p50 (NF-κB p50) as the model, the limit of detection was low to 0.14 nM. Furthermore, the sequence-specific recognition of NF-κB p50 with DNA displayed excellent selectivity and specificity. The results in present work showed that this strategy will be a promising tool for DNA-binding proteins analysis in biomedical exploration and clinical diagnosis.