Kinetically Programmed Signaling Cascades for Molecular Detection

Upon external stimulation, living cells activate a series of precisely programmed signaling cascade reactions that efficiently convert multiple stimuli into relevant output signals, thereby maintaining physiological homeostasis. These naturally evolved molecular networks have recently inspired the development of new chemical strategies in fields ranging from synthetic biology to molecular computing, drug delivery, and biosensing systems. While some studies have begun to explore how programming the thermodynamics of these bioinspired chemical systems can optimize their performance, the impact of programming their kinetics remains largely unexplored. Here, we leveraged the modularity and programmability of DNA chemistry to develop a simple DNA-based signaling cascade to measure the concentration of specific molecules and investigated how programming its kinetics affects its performance. This signaling cascade comprises four modules (input, receptor, processor, and output) and three molecular interactions for which we have characterized all intrinsic rate constants. Through simulations and experiments, we demonstrated that careful kinetic programming can significantly enhance the rate, gain, and sensitivity of the signaling cascade output. We further illustrated the versatility and modularity of this cascade by adapting it for the detection of four different molecules ( small molecules and proteins). We also showed that it can be readily adapted into a rapid, one-step, inexpensive electrochemical sensor enabling therapeutic drug monitoring (TDM) at home directly from a drop of blood. We believe that similar kinetically programmed signaling cascades could be developed for a wide range of chemical applications, allowing complex, multistep workflows to be streamlined into rapid, single-step reactions.