Toyonari Masato, Aso Kaori, Nakakuki Takashi
Department of Intelligent and Control Systems, Faculty of Computer Science and Systems Engineering, Kyushu Institute of Technology, 680-4 Kawazu, Iizuka 820-8502, Fukuoka, Japan.
Micromachines (Basel). 2024 Nov 30;15(12):1466. doi: 10.3390/mi15121466.
DNA chemical reaction networks can perform complex information processing through careful design of reaction kinetics, which involves the reaction network structure, rate constants, and initial concentrations. The toehold-mediated strand displacement reaction (TMSDR) is a key mechanism in creating DNA circuits, offering a rational design approach by integrating individually designed TMSDRs. Tools such as VisualDSD and NUPACK facilitate the efficient design of these systems by allowing precise tuning of reaction parameters. However, discrepancies between simulated and experimental results can occur, often due to the modification of reporter molecules. Recently, fluorophore dyes and quenchers were found to significantly impact the dynamics of irreversible TMSDRs, altering them by nearly two orders of magnitude. The impact on reaction dynamics varies with the modification site of these reporters. This study examines the mechanisms of reporter modifications affecting reversible TMSDRs, influencing transient and steady-state characteristics. This is crucial for DNA circuit design, which integrates reversible and irreversible TMSDRs. Our findings indicate that modifying fluorescent dye and quencher an appropriate distance apart (e.g., toehold length) can minimize adverse effects on the DNA reaction dynamics while ensuring effective FRET, therefore improving the accuracy of experimental verification for DNA reaction systems.
DNA化学反应网络可以通过精心设计反应动力学来执行复杂的信息处理，这涉及反应网络结构、速率常数和初始浓度。托脚介导的链置换反应（TMSDR）是创建DNA电路的关键机制，通过整合单独设计的TMSDR提供了一种合理的设计方法。VisualDSD和NUPACK等工具通过允许精确调整反应参数，促进了这些系统的高效设计。然而，模拟结果和实验结果之间可能会出现差异，这通常是由于报告分子的修饰所致。最近发现，荧光团染料和猝灭剂会显著影响不可逆TMSDR的动力学，使其改变近两个数量级。对反应动力学的影响随这些报告分子的修饰位点而变化。本研究探讨了影响可逆TMSDR的报告分子修饰机制，及其对瞬态和稳态特征的影响。这对于整合可逆和不可逆TMSDR的DNA电路设计至关重要。我们的研究结果表明，将荧光染料和猝灭剂修饰在适当的距离（例如，托脚长度）可以在确保有效荧光共振能量转移（FRET）的同时，将对DNA反应动力学的不利影响降至最低，从而提高DNA反应系统实验验证的准确性。
