Self-phosphorylating DNAzyme DK1 enables programmable multi-analyte readout via PfAgo.

DNAzymes possessing kinase-like activities have long held theoretical promise, yet their practical implementation has remained significantly limited. Notably, DNAzyme kinase 1 (DK1), discovered over two decades ago, exhibits a unique self-phosphorylation capability upon encountering specific substrates like ATP, but its broad-based and programmable applications have not yet been fully realized. In this study, we innovatively couple DK1's autophosphorylation mechanism with the PfAgo to establish a novel programmable cascade sensing platform named RASTEN (Robust pfAgo-based Strategy for POC Testing Non-nucleic Acid and Nucleic Acid). Upon activation by ATP-induced DK1 self-phosphorylation, RASTEN leverages PfAgo to initiate a sequential cleavage of two reporter probes, enabling rapid, one-pot, visual detection of diverse biomolecular targets. Utilizing this DK1-PfAgo cascade system, we successfully developed a practical point-of-care (POC) diagnostic assay using test strips, demonstrated through highly sensitive detection of ATP in simulated human serum samples. Additionally, RASTEN exhibits exceptional versatility by harnessing alkaline phosphatase (ALP) activity as another example, effectively achieving sensitive visual detection of protein biomarkers. The inherent programmability of RASTEN, facilitated by the modifiable DK1 substrate sequences, further broadens its applicability across various nucleic acid and non-nucleic acid targets. Clinical validation highlights RASTEN's substantial advantages over commercial diagnostic kits, including significantly reduced costs, streamlined user experience, and instrument-free operation. This innovative integration of DK1 and PfAgo establishes a robust and adaptable biosensing platform, presenting compelling opportunities for versatile, cost-effective, and practical multi-analyte diagnostics.