Endonuclease IV-Regulated DNAzyme Motor for Universal Single-nucleotide Variation Discrimination

Single-nucleotide variation (SNV) detection plays significant roles in disease diagnosis and treatment. Generally, auxiliary probe, restricted design rules, complicated detection system, and repeated experimental parameter optimization are needed to obtain satisfactory tradeoff between sensitivity and selectivity for SNV discrimination, especially when different mutant sites need to be distinguished. To overcome these limitations, we developed a universal, straightforward, and relatively cheap SNV discrimination strategy, which simultaneously possessed high sensitivity and selectivity. The excellent performance of this strategy was ascribed to the SNV discrimination property of endonuclease IV (Endo IV) and the different hydrolysis behavior between free deoxyribozyme (DNAzyme) and the trapped DNAzyme to the substrates modified on gold nanoparticles (AuNPs). When Endo IV recognized the mutant-type target (MT), free DNAzyme was released from the probe, and the DNAzyme motor was activated with the help of cofactor Mn²⁺ to generate an amplified fluorescence signal. On the contrary, the wild-type target (WT) could not effectively trigger the DNAzyme motor. Moreover, for different SNV types, the corresponding probe could be designed by simply changing the sequence hybridized with the target and retaining the DNAzyme sequence. Thus, the fluorescence signal generation system does not need to change for different SNV targets. Five clinical-related SNVs were determined with the limit of detection (LOD) ranging from 0.01 to 0.05%, which exhibited competitive sensitivity over existing SNV detection methods. This strategy provided another insight into the properties of Endo IV and DNAzyme, expanded the applications of DNAzyme motor, and has great potential to be used for precision medicine.