David Y. Zhang

Biological organisms perform complex information processing and control tasks using sophisticated biochemical circuits, yet the engineering of such circuits remains ineffective compared with that of electronic circuits. To systematically create complex yet reliable circuits, electrical engineers use digital logic, wherein gates and subcircuits are composed modularly and signal restoration prevents signal degradation. We report the design and experimental implementation of DNA-based digital logic circuits. We demonstrate AND, OR, and NOT gates, signal restoration, amplification, feedback, and cascading. Gate design and circuit construction is modular. The gates use single-stranded nucleic acids as inputs and outputs, and the mechanism relies exclusively on sequence recognition and strand displacement. Biological nucleic acids such as microRNAs can serve as inputs, suggesting applications in biotechnology and bioengineering.

DNA is increasingly being used as the engineering material of choice for the construction of nanoscale circuits, structures, and motors. Many of these enzyme-free constructions function by DNA strand displacement reactions. The kinetics of strand displacement can be modulated by toeholds, short single-stranded segments of DNA that colocalize reactant DNA molecules. Recently, the toehold exchange process was introduced as a method for designing fast and reversible strand displacement reactions. Here, we characterize the kinetics of DNA toehold exchange and model it as a three-step process. This model is simple and quantitatively predicts the kinetics of 85 different strand displacement reactions from the DNA sequences. Furthermore, we use toehold exchange to construct a simple catalytic reaction. This work improves the understanding of the kinetics of nucleic acid reactions and will be useful in the rational design of dynamic DNA and RNA circuits and nanodevices.

Artificial biochemical circuits are likely to play as large a role in biological engineering as electrical circuits have played in the engineering of electromechanical devices. Toward that end, nucleic acids provide a designable substrate for the regulation of biochemical reactions. However, it has been difficult to incorporate signal amplification components. We introduce a design strategy that allows a specified input oligonucleotide to catalyze the release of a specified output oligonucleotide, which in turn can serve as a catalyst for other reactions. This reaction, which is driven forward by the configurational entropy of the released molecule, provides an amplifying circuit element that is simple, fast, modular, composable, and robust. We have constructed and characterized several circuits that amplify nucleic acid signals, including a feedforward cascade with quadratic kinetics and a positive feedback circuit with exponential growth kinetics.