Leveraging Steric Moieties for Kinetic Control of DNA Strand Displacement Reactions

Abstract

DNA strand displacement networks are a critical part of dynamic DNA nanotechnology and are proven primitives for implementing chemical reaction networks. Precise kinetic control of these networks is important for their use in a range of applications. Among the better understood and widely leveraged kinetic properties of these networks are toehold sequence, length, composition, and location. While steric hindrance has been recognized as an important factor in such systems, a clear understanding of its impact and role is lacking. Here, a systematic investigation of steric hindrance within a DNA toehold-mediated strand displacement network was performed through tracking kinetic reactions of reporter complexes with incremental concatenation of steric moieties near the toehold. Two subsets of steric moieties were tested with systematic variation of structures and reaction conditions to isolate sterics from electrostatics. Thermodynamic and coarse-grained computational modeling was performed to gain further insight into the impacts of steric hindrance. Steric factors yielded up to 3 orders of magnitude decrease in the reaction rate constant. This pronounced effect demonstrates that steric moieties can be a powerful tool for kinetic control in strand displacement networks while also being more broadly informative of DNA structural assembly in both DNA-based therapeutic and diagnostic applications that possess elements of steric hindrance through DNA functionalization with an assortment of chemistries.

Plug-and-Play Module for Reversible and Continuous Control of DNA Strand Displacement Kinetics

“…3 Modeling and controlling the kinetics of TMSD have long been a central focus of DNA nanotechnology, leading to the development of a variety of theoretical tools, such as OxDNA, 20−22 and control modules, including toeholdexchange, 23 remote toehold, 24 allosteric toehold, 25 mutationbased kinetic regulation, 26,27 and steric moieties. 28 So far, all control modules are hardwired into TMSD, which is irreversible and difficult to integrate into existing DNA-based CRNs. For example, remote toehold achieves continuous tuning of TMSD kinetics but must introduce varying lengths of spacer domains between the toehold and branch migration domains.…”

“…Toehold-mediated DNA strand displacement (TMSD) is one of the most widely used building blocks in creating dynamic and dissipative DNA-based CRNs (Figure a) . Modeling and controlling the kinetics of TMSD have long been a central focus of DNA nanotechnology, leading to the development of a variety of theoretical tools, such as OxDNA, − and control modules, including toehold-exchange, remote toehold, allosteric toehold, mutation-based kinetic regulation, , and steric moieties . So far, all control modules are hardwired into TMSD, which is irreversible and difficult to integrate into existing DNA-based CRNs.…”

“…For example, remote toehold achieves continuous tuning of TMSD kinetics but must introduce varying lengths of spacer domains between the toehold and branch migration domains . Allosteric toehold, mutation-based, and steric control approaches also need to reprogram sequences for TMSD by including additional domains or mutations for kinetic regulation. − Toward the ongoing needs for devising out-of-equilibrium CRNs and temporal control of existing CRNs, an ideal kinetic control module shall meet at least three criteria, including (1) being an “add-on” module that can be readily and flexibly integrated into existing DNA-based CRNs without the need for reprograming sequences of TMSD; (2) reversible with kinetic control module that can be activated and deactivated in real time; and (3) capable of fine-grained and continuous kinetic tuning by altering practically manipulable parameters, such as concentration. Herein, we introduce antitoehold, a plug-and-play module that for the first time meets all three criteria and allows reversible and continuous control of TMSD kinetics.…”

Importance of DNA nanotechnology for DNA methyltransferases in biosensing assays

“…68 Additionally, DNA nanomaterials have been used in gene therapy and high-contrast imaging. 69 These are the applications of new methods and tools for early disease diagnosis and treatment. The future of DNA nanomaterials in biomedicine remains promising as the technology continues to advance.…”

Rationally Designed Dual Base Pair Mismatch Enables Toehold-Mediated Strand Displacement to Efficiently Recognize Single-Nucleotide Polymorphism without Enzymes

“…In recent years, benefiting from low-cost synthesis and purification of DNA, superior programmability, and good biocompatibility, − DNA-based nonenzymatic biosensors have received considerable concern in biological detection. DNA gains more and more attention as a biomolecular engineering building material to replace proteases. , TMSD is a technique, in which the target DNA binds to the exposed toehold domain of double-stranded DNA with the help of a skillfully designed toehold domain and then triggers the chain replacement reaction to release the complementary single-stranded DNA. − Currently, TMSD reactions have become a relatively mature technique for constructing enzyme-free detection systems, and depending on this technology, different sequences of DNA or RNA with concentrations as low as femtomolar or even attomolar level can be accurately detected. − However, as most researchers often study, TMSD is mostly used for specific recognition between different sequences but does not have accurate resolution for a single base in a sequence. Therefore, the TMSD reaction cannot be well used to specifically detect single base mutations.…”