A Logical Molecular Circuit for Programmable and Autonomous Regulation of Protein Activity Using DNA Aptamer–Protein Interactions

Abstract

Researchers increasingly envision an important role for artificial biochemical circuits in biological engineering, much like electrical circuits in electrical engineering. Similar to electrical circuits, which control electromechanical devices, biochemical circuits could be utilized as a type of servomechanism to control nanodevices in vitro, monitor chemical reactions in situ, or regulate gene expressions in vivo.1 As a consequence of their relative robustness and potential applicability for controlling a wide range of in vitro chemistries, synthetic cell-free biochemical circuits promise to be useful in manipulating the functions of biological molecules. Here we describe the first logical circuit based on DNA-protein interactions with accurate threshold control, enabling autonomous, self-sustained and programmable manipulation of protein activity in vitro. Similar circuits made previously were based primarily on DNA hybridization and strand displacement reactions. This new design uses the diverse nucleic acid interactions with proteins. The circuit can precisely sense the local enzymatic environment, such as the concentration of thrombin, and when it is excessively high, a coagulation inhibitor is automatically released by a concentration-adjusted circuit module. To demonstrate the programmable and autonomous modulation, a molecular circuit with different threshold concentrations of thrombin was tested as a proof of principle. In the future, owing to tunable regulation, design modularity and target specificity, this prototype could lead to the development of novel DNA biochemical circuits to control the delivery of aptamer-based drugs in smart and personalized medicine, providing a more efficient and safer therapeutic strategy.

DNA-Based Enzyme Reactors and Systems

“…The circuit by Han et al [55] and its working principle are depicted in Figure 4d. The enzyme α-thrombin is a protease that aids blood coagulation by converting fibrinogen to fibrin.…”

A survey of advancements in nucleic acid-based logic gates and computing for applications in biotechnology and biomedicine

“…developed a logical molecular circuit that can realize programmable and autonomous manipulation of thrombin function via a set threshold. 78 As shown in Figure 7A, the process includes three stages. In the Input Convertor , upon addition of thrombin, TA-29 binds to its target position and releases the partially complementary strand as DNA-input.…”

“…Three modules are involved: Input Convertor, Threshold Controller and Inhibitor Generator (reprinted by permission from reference [78], Copyright 2012, American Chemical Society). (B) Connection of AND and NOR logic gates using protein as output.…”

Molecular Engineering to Enhance Aptamer Functionality

“…5.7) [56]. In general, a programmable and autonomous circuit with threshold control is constructed of three DNA modules: an input convertor that converts the protein input to DNA input for downstream cascade reactions; a threshold controller that sets the threshold concentration for the system to maintain regular protein activity; and an inhibitor generator that inhibits excessively high protein activity once it surpasses the threshold.…”