Allosteric DNAzyme for sensitive detection of nucleic acids for molecular diagnosis

Abstract Nucleic acids in biofluids are emerging biomarkers for molecular diagnosis of diseases, whose clinical use has been hindered by the lack of sensitive and convenient detection assays. Herein, we report a sensitive nucleic acid detection method based on allosteric DNAzyme biosensors named SPOT ( s ensitive loo p -initiated DNAzyme biosens o r for nucleic acid detection) by rationally designing a programmable DNAzyme of endonuclease capability. SPOT can be activated once a nucleic acid target of specific sequence binds to its allosteric module to induce conformational reconfiguration of DNAzyme enabling continuous cleavage of molecular reporters. SPOT provides a highly robust platform for sensitive (LOD: femtomolar for miRNAs, attomolar for SARS-CoV-2 RNA), specific (single-nucleotide discrimination), and convenient (one-step, one-pot, preamplification-free) detection of low-abundant nucleic acid biomarkers. For clinical validation, we demonstrated that SPOT is capable of detecting serum miRNAs (e.g., miR-155, miR-21) from patients for the precise diagnosis of breast cancer, gastric cancer, and prostate cancer. Furthermore, SPOT exhibits potent detection capability over SARS-CoV-2 RNA from clinical swabs with high sensitivity and specificity. Lastly, SPOT is compatible with point-of-care testing modalities such as lateral flow assay to enable convenient visualization. Hence, we envision that SPOT may serve as a robust platform for sensitive detection of a variety of nucleic acid targets towards clinical applications in molecular diagnosis.