Improving dynamic control of toehold-mediated strand displacement reactions through phosphorothioate modifications

The toehold-mediated strand displacement reaction plays a crucial role in the functionality of DNA nanodevices. However, the reaction's velocity is heavily reliant on the length of the toehold region. A shorter toehold can impede the speed and completeness of the reaction. This study utilized DNA phosphorothioate modification to analyze its impact on these significant processes. The findings revealed that a shorter toehold (Toehold < 5 nt) accelerates the initial strand release, thus expediting the entire reaction when modified with phosphorothioate. Conversely, a longer toehold (Toehold > 4 nt) with phosphorothioate modification enhances the displacement reaction's extent. Furthermore, this newfound knowledge was applied to DNA-based reaction systems such as catalytic hairpin assembly and DNA probabilistic circuit, demonstrating that phosphorothioate usage can effectively regulate and enhance the functionality of these systems. This development holds promise for the future advancements in biocomputing, nanotechnology, and gene therapy. The discovery for the first time that phosphorothioate modifications can enhance the rate of TMSD in the short toehold region. Furthermore, we applied our findings to CHA circuits and DNA probabilistic circuits.