Rational Engineering of a Dynamic, Enzyme-Driven DNA Walker for Intracellular Dual-Enzyme Activity Sequentially Monitoring and Imaging

Monitoring enzyme activity is crucial in both scientific research and clinical applications. However, abnormalities in a single enzyme's activity can indicate multiple diseases, limiting the specificity of single enzyme activity monitoring in clinical diagnosis. We developed a dynamic DNA walker that can be sequentially activated by two enzymes, enabling the monitoring and imaging of both enzyme activities within cells. Initially, the DNA walker contains a site for apurinic/apyrimidinic endonuclease 1 (APE1). Upon APE1 activation, the DNA walker forms specific structures recognized and cleaved by Flap endonuclease 1 (FEN1). The temporal disparity between the activities of APE1 and FEN1 allows for the sequential monitoring and imaging of both enzymes, reducing the likelihood of false-positive results. To enhance local concentration and decrease reaction time, the DNA walk sequence was attached to the surface of gold nanoparticles (AuNPs). The fruition of this endeavor will facilitate the investigation and advancement of multiple enzyme activity monitoring and imaging methods and technologies, while simultaneously broadening the domains of application for DNA nanotechnology.