Control of Reversible Formation and Dispersion of the Three Enzyme Networks Integrating DNA Computing

The majority of biological reactions in the cytoplasm of living cells occur via enzymatic cascade reactions. To achieve efficient enzyme cascade reactions mimicking the proximity conditions of enzymes in the cytoplasm, the proximity of each enzyme, creating a high local concentration of proteins, has been recently investigated using the conjugation of synthetic polymer molecules, proteins, and nucleic acids. Although there have been methodologies reported for the complex formation and enhanced activity of cascade reactions due to the proximity of each enzyme using DNA nanotechnology, one pair of the enzyme (GOx and HRP) complex is only assembled by the mutual independence of various shapes of the DNA structure. This study reports the network formation of three enzyme complexes assembled by a triple-branched DNA structure as a unit, thus enabling the reversible formation and dispersion of the three enzyme complex networks using single-stranded DNA, RNA, and enzymes. It was found that the activities of the three enzyme cascade reactions in the enzyme–DNA complex network were controlled by formation and dispersion of the three enzyme complex networks, due to the proximity of each enzyme with the enzyme–DNA complex network. Furthermore, three micro RNA sequences for breast cancer biomarkers were successfully detected using an enzyme–DNA complex network integrated with DNA computing. Overall, the reversible formation and dispersion of the enzyme–DNA complex network through the external stimulation of biomolecules and DNA computing provide a novel platform for controlling the production amount, diagnosis, theranostics, and biological or environmental sensing.