Electrical and Computer Engineering, University of Texas at Austin, Austin, Texas 78712, United States.
Paul G. Allen School of Computer Science and Engineering, University of Washington, Seattle, Washington 98195, United States.
ACS Synth Biol. 2023 Apr 21;12(4):993-1006. doi: 10.1021/acssynbio.2c00356. Epub 2023 Apr 4.
Molecular control circuits embedded within chemical systems to direct molecular events have transformative applications in synthetic biology, medicine, and other fields. However, it is challenging to understand the collective behavior of components due to the combinatorial complexity of possible interactions. Some of the largest engineered molecular systems to date have been constructed using DNA strand displacement reactions, in which signals can be propagated without a net change in base pairs (enthalpy neutral). This flexible and programmable component has been used for constructing molecular logic circuits, smart structures and devices, for systems with complex autonomously generated dynamics, and for diagnostics. Limiting their utility, however, strand displacement systems are susceptible to the spurious release of output in the absence of the proper combination of inputs (leak), as well as reversible unproductive binding (toehold occlusion) and spurious displacement that slow down desired kinetics. We systematize the properties of the simplest enthalpy-neutral strand displacement cascades (logically linear topology), and develop a taxonomy for the desired and undesired properties affecting speed and correctness, and trade-offs between them based on a few fundamental parameters. We also show that enthalpy-neutral linear cascades can be engineered with stronger thermodynamic guarantees to leak than non-enthalpy-neutral designs. We confirm our theoretical analysis with laboratory experiments comparing the properties of different design parameters. Our method of tackling the combinatorial complexity using mathematical proofs can guide the engineering of robust and efficient molecular algorithms.
嵌入化学系统以指导分子事件的分子控制回路在合成生物学、医学和其他领域具有变革性的应用。然而，由于可能的相互作用的组合复杂性，理解组件的集体行为具有挑战性。迄今为止，一些最大的工程分子系统是使用 DNA 链置换反应构建的，其中信号可以在不改变碱基对（焓中性）的情况下传播。这种灵活且可编程的组件已用于构建分子逻辑电路、智能结构和设备、具有复杂自主生成动力学的系统以及用于诊断。然而，限制它们的应用的是，链置换系统容易在没有适当输入组合（泄漏）的情况下错误地释放输出，以及可逆的非生产性结合（趾部闭塞）和虚假置换，从而减缓所需的动力学。我们系统地研究了最简单的焓中性链置换级联（逻辑线性拓扑）的特性，并根据几个基本参数为影响速度和正确性的所需和不希望的特性以及它们之间的权衡开发了一个分类法。我们还表明，焓中性线性级联可以用比非焓中性设计更强的热力学保证来设计泄漏。我们通过比较不同设计参数的实验室实验来验证我们的理论分析。我们使用数学证明来解决组合复杂性的方法可以指导稳健高效的分子算法的工程设计。
