Rational Engineering\nof a Dynamic, Enzyme-Driven DNA\nWalker for Intracellular Dual-Enzyme Activity Sequentially Monitoring\nand Imaging

Monitoring enzyme activity is crucial in both scientific\nresearch\nand clinical applications. However, abnormalities in a single enzyme’s\nactivity can indicate multiple diseases, limiting the specificity\nof single enzyme activity monitoring in clinical diagnosis. We developed\na dynamic DNA walker that can be sequentially activated by two enzymes,\nenabling the monitoring and imaging of both enzyme activities within\ncells. Initially, the DNA walker contains a site for apurinic/apyrimidinic\nendonuclease 1 (APE1). Upon APE1 activation, the DNA walker forms\nspecific structures recognized and cleaved by Flap endonuclease 1\n(FEN1). The temporal disparity between the activities of APE1 and\nFEN1 allows for the sequential monitoring and imaging of both enzymes,\nreducing the likelihood of false-positive results. To enhance local\nconcentration and decrease reaction time, the DNA walk sequence was\nattached to the surface of gold nanoparticles (AuNPs). The fruition\nof this endeavor will facilitate the investigation and advancement\nof multiple enzyme activity monitoring and imaging methods and technologies,\nwhile simultaneously broadening the domains of application for DNA\nnanotechnology.