Enzyme-Triggered DNA Sensor Technology for Spatially-Controlled, Cell-Selective Molecular Imaging.

Abstract

ConspectusWith unparalleled programmability, DNA has evolved as a powerful scaffold for engineering intricate and dynamic systems that can perform diverse tasks. By allowing serial detection of molecular targets in complex cellular milieus, increasingly sophisticated DNA sensors have not only promoted significant advances in unveiling the fundamental mechanisms of various pathophysiological processes but also provided a useful toolkit for disease diagnostics based on molecular signatures. Despite much progress, an inherent limitation of DNA-based sensors is that they often lack spatial control and cell-type selectivity for the sensing activity because of their "always active" design mechanism. Since most molecular targets of interests are not exclusive to disease cells, they are also shared by normal cells, the application of such biosensors for disease-specific imaging is limited by inadequate signal-to-background ratios due to indistinguishable signal response in both disease and normal cells. Therefore, imparting biosensors with spatial controllability remains a key issue to achieve molecular imaging with high sensitivity and cell specificity.As a biocatalyst, enzyme has been found to be closely related with the pathological conditions of numerous diseases. For example, many nucleases, protease, and kinases have been identified overexpressed in disease cells and considered as important biomarkers of cancer, inflammation, and neurological diseases. Recently, we have envisioned that such pathophysiology-associated enzymes could be leveraged as endogenous triggers to achieve spatial control over the molecular imaging activity of the DNA-based sensors with improved cell-specificity. In this Account, we outline the research efforts from our group on the development of endogenous enzyme-triggered, DNA-based sensor technology that enables spatially controlled, cell-type selective molecular imaging. With programmable DNA design and further engineering of enzymatically cleavable sites, a series of DNAzyme- and aptamer-based sensors have been developed for enzyme-controlled imaging of various molecular targets (e.g., metal ions and small molecules) in a cancer cell-selective manner. In particular, by introduction of PNA as bridge molecules to engineer DNA-based sensors with functional peptides, the conceptual design of protease-activated DNA biosensors has been established for spatioselective molecular imaging in cancer cells and extracellular tumor microenvironments. Furthermore, enzyme-triggered signal amplification approaches, such as enzymatically activated molecular beacon and catalytic hairpin assembly, have been developed for spatially selective RNA imaging in specific disease cells (e.g., inflammatory cells and cancer cells), which enables enhanced disease-site specificity and thus improved signal-to-background ratio. The signal amplification strategy is further expanded to cell-selective amplified imaging of non-RNA species through the combination with functional DNA design. Finally, the challenges and potential future directions in this burgeoning field are discussed. We hope this Account offers insights into rational design of enzymatically controlled, DNA-based sensor platforms for opening new frontiers in spatially resolved, cell-selective molecular imaging. We believe that the continuing advances in DNA-based molecular sensing technology together with the discoveries of diverse disease-associated enzymes will promise to usher a new era of diagnosis.