Self-Phosphorylating Deoxyribozyme Initiated Cascade Enzymatic Amplification for Guanosine-5′-triphosphate Detection

The self-phosphorylating deoxyribozymes identified by in vitro selection can catalyze their own phosphorylation by utilizing phosphate donor guanosine-5'-triphosphate (GTP) which plays a critical role in a majority of cellular processes. On the basis of the unique properties of self-phosphorylating deoxyribozymes, we report a novel GTP sensor coupled with lambda exonuclease cleavage reaction and nicking enzyme assisted fluorescence signal amplification process. The deoxyribozymes with special catalytic and structural characteristics display good stability compared to protein and RNA enzymes. We combined these properties with enzymatic recycling cleavage strategy to build a sensor which produced enhanced fluorescence signal. Sensitive and selective detection of GTP was successfully realized with the well-designed deoxyribozyme-based sensing platform by taking advantage of the self-phosphorylating ability of the kinase deoxyribozyme, efficient digestion capacity of lambda exonuclease, and enzymatic recycling amplification of nicking enzyme. The method not only provides a platform for detecting GTP but also shows great potential in analyzing a variety of targets by combining deoxyribozymes with signal amplification strategy.