Design And Application Of A Pistol-like DNAzyme In Sugar Sensors

In the early 1980 s, Cech and Altman found RNA molecules with catalytic functions in introns and RNase P respectively, called RNAzyme(ribozyme). The discovery of ribozymes changed the traditional concept that “all enzymes are proteins”, sparking the study of catalytic DNAs. In 1994, Breaker and Joyce for the first time reported a Pb2+ dependent RNA-cleaving DNA molecule and its kinetics reaction followed the Michaelis-Menten equation. Such catalytic DNA with high specificity is known as DNAzyme(deoxyribozyme). So far, there is no naturally occurring DNAzymes in living bodies and all DNAzymes are obtained by in vitro selection. The majority of DNAzymes perform nucleic acid cleavage reactions, such as pistol-like DNAzyme(PLDz). This DNAzyme with self-cleaving activity was developed in the Breaker laboratory through in vitro selection using "Cu2+ /Vitamin C" as cofactors. Its catalytic mechanism is thought to be a radical-mediated oxidative DNA cleavage reaction. Based on the studies of Breaker and others, we have explored the pistol-like DNAzyme from its enzymatic properties, conformation-function relationships and catalytic mechanism. This article will focus on researches of a glucose sensor and a sucrose sensor based on the pistol-like DNAzyme.We designed a dual-enzyme coupled glucose sensor GOx-PLDz 1.0, based on the relationship between PLDz’s catalytic function and its cofactors. Its working principle is: β-D-glucose in samples as a first signal can be recognized and catalyzed by GOx forming gluconic acid and H2O2. The generated H2O2 as a second signal can be further identified by PLDz, which performs a self-cleavage reaction in the presence of H2O2 and divalent metal ions. The slope of the cleavage rate of PLDz versus glucose concentration curve was fitted with a "Double Boltzmann" equation. The detection limit of GOx-PLDz 1.0 is 5 μM, and it has been applied for glucose detection in tears and saliva, the result is 720±81 and 405±56 μM respectively, achieving a non-invasive way to detect glucose levels. On the basis of GOx-PLDz 1.0, we introduced a trans pistollike DNAzyme to build GOx-PLDz 1.1, showing that the trans pistol-like DNAzyme can be used as a new generation of glucose sensor element.To improve the availability and operability of glucose sensor, we developed a highly sensitive fluorescent glucose sensor GOx-PLDz 2.0 on the basis of GOx-PLDz 1.0 and 1.1. Detection principle of GOx-PLDz 2.0 is the same as GOx-PLDz 1.0 and 1.1, the most difference between them is the signal output section. We modified pistol-like DNAzyme and it substrate using fluorescent/quencher molecules. In the presence of glucose, GOx-PLDz 2.0 emitted fluorescence signal, which can significantly increase the detection sensitivity of the sensor. The relationship between the fluorescent signal intensity and the glucose concentration was fitted with "Extreme" equation and the detection limit of GOx-PLDz 2.0 is 50 nM. We have applied GOx-PLDz 2.0 sensor to detect and analyze 10 saliva samples.In the study of conformation-function relationship in pistol-like DNAzyme, we obtained a mutant PLDz-T3 G with increased activity. We designed a three-enzymecoupled sucrose sensor IGP1.0 using the combination of invertase(INV), glucose oxidase(GOx) and a mutant of pistol-like DNAzyme(PLDz-T3G), based on the glucose sensor GOx-PLDz 1.0. Its detection limit is 5 μM sucrose. With the pretreatment of GOx, IGP 1.0 sensor showed strong anti-interference. We applied IGP 1.0 to test sucrose concentrations in eight kinds of drinks. We found that Coca-Cola Zero contains only trace amounts of sucrose; the sucrose levels contained in Coca-Cola, Pepsi and Red Bull are in the range as in the ingredients lists; but the tea beverage such as green tea interfere with sucrose detection due to vitamin C within it.Based on pistol-like DNAzyme, we have successfully designed glucose sensors GOx-PLDz 1.0, 1.1 and 2.0 as well as a sucrose sensor IGP 1.0, indicating that DNAzymes have broader application prospects in sugar sensors. Based on this, we can develop "super-sensitive", "visualization" glucose and sucrose sensors in the future by combination with other functional materials, such as gold nanoparticles, carbon nanotubes or graphene. The former one serves the majority of patients with diabetes and vulnerable populations to monitor daily glucose levels, the latter one is used to study sucrose metabolism in vivo.