Junction toehold: A novel approach for flexible DNA strand displacement and bioassay

Toehold-mediated DNA strand displacement is a simple yet powerful approach to design various DNA devices, and also receives more and more attention for bio-assay applications. It is critical to activate toehold in a programmable and controllable way to regulate DNA strand displacement. Thus it is desirable to develop new toehold to promote the programmability and versatility of DNA devices, and enrich its bio-assay applications. Here a novel junction toehold is reported with low signal leakage based on a junction motif. The DNA strand displacement process is cooperatively and sequentially activated by two inputs with independent oligonucleotide sequences. The rate constants of individual reaction steps were characterized in detail, and the kinetics can be flexibly tuned independently by multi-parameters. Because of the diverse reaction sites in the junction toehold, we are able to construct various DNA devices with advanced architecture including a concatenated DNA circuit, a parallel-activated device and so on. Moreover, the activity of ribonuclease or endonuclease was sensitively and selectively assayed via enzyme-activated strand displacement method, demonstrating the power of junction toehold for strand displacement regulation and bio-assay.