Nucleic acid analysis plays an important role indiagnosing diseases as well as understanding biology. Despite advancesin technology, there is still a need to develop a rapid and simplemethod to detect specific nucleic acids, especially in remote locationsand low-resource cases. Here, we proposed a proximity proteolysisreaction in which the reaction between protease and zymogen isenhanced in the presence of a target molecule. The pair of proteins wassite-specifically modified with oligonucleotides, and the conjugateswere used to develop a method of detecting nucleic acids. Target DNAand RNA could be detected in less than 1 h at sub-nanomolarconcentrations based on an absorbance signal. The assay method wasresistant to interference by biological matrixes, and its sensitivity couldbe improved when combined with an isothermal nucleic acidamplification method. The results demonstrated the feasibility of this proximity proteolysis reaction as a new platformtechnology for detecting specific nucleic acid sequences.
In this study, the concept of autoinhibition, a mechanism of proteinactivity regulation, was applied to the design and engineering of ab-lactamase zymogen. Using this zymogen, a sensitive proteaseassay method was developed in which activation of the zymogen byproteases produces an amplified absorbance signal. The approachreported here can be adapted for engineering of zymogens asbiological sensors and components of synthetic signaling pathways.
Department of Molecular Science and Technology, Ajou University
206 Worldcup-ro, Yeongtong-gu, Suwon, 16499 Korea