SHF: Small: High-speed DNA polymerase CRNs for signal amplification, oscillation, consensus, and linear control

SHF：小型：高速 DNA 聚合酶 CRN，用于信号放大、振荡、一致性和线性控制

基本信息

批准号：
2113941
负责人：
John Reif
金额：
$ 50万
依托单位：
Duke University
依托单位国家：
美国
项目类别：
Standard Grant
财政年份：
2021
资助国家：
美国
起止时间：
2021-10-01 至 2024-09-30
项目状态：
已结题

来源：
https://www.nsf.gov/awardsearch/showAward?AWD_ID=2113941&HistoricalAwards=false
关键词：
SHF Small speed DNA polymerase

项目摘要

The regulation of cellular and molecular processes typically involves complex biochemical processes, which are termed chemical reaction networks (CRNs). Synthetic CRNs using reactions on DNA molecules can be systematically designed to approximate sophisticated biochemical processes. However, most of the prior experimental protocols for CRNs relied on either DNA strand-displacement hybridization or enzymatic reactions, and the resulting synthetic systems usually suffer from either slow rates or leaky reactions. In contrast, much higher reaction rates can be obtained by the DNA enzyme strand-displacement polymerase (PSD). This project is investigating a wide variety of fast and robust CRNs using only DNA hybridization and PSD reactions. The project includes design, simulation, and experimental implementations. Further, the project is highly interdisciplinary and impacts interdisciplinary education at undergraduate and graduate levels. The project engages students (with emphasis on women and under-represented minorities) from different academic levels across multiple disciplines in mentoring and teaching. Hands-on demonstrations of DNA computing and CRNs are being designed for outreach programs at Duke and local high schools. Workshops and lectures are disseminating knowledge of advanced PSD-based nanoscience concepts to undergraduate and graduate student audiences. The regulation of cellular and molecular processes typically involves complex biochemical processes, which are termed chemical reaction networks (CRNs). Synthetic CRNs using reactions on DNA molecules can be systematically designed to approximate sophisticated biochemical processes. However, most of the prior experimental protocols for CRNs relied on either DNA strand-displacement hybridization or enzymatic reactions, and the resulting synthetic systems usually suffer from either slow rates or leaky reactions. In contrast, much higher reactions rates can be obtained by the DNA enzyme strand-displacement polymerase (PSD). This project is investigating a wide variety of fast and robust CRNs using only DNA hybridization and PSD reactions. The project includes design, simulation, and experimental implementations. The key CRNs investigated in this project include (i) an autocatalytic amplifier, (ii) a dynamic oscillatory system, (iii) a molecular-scale consensus protocol, and (iv) a linear control system. All of these CRNs have important practical applications. The project has already completed the design, simulation and preliminary experiments of some simple CRNs using PSD, including in silico demonstration of dynamic CRNs using PSD, which provided estimates of reaction rate, leak, and false positives for these simple CRNs. Further, the project has already completed the design, simulation & experimental demonstration of dynamic oscillatory CRN systems using PSD to identify the number of cycles, precision of cycles, sensitivity to initial concentrations and approximation to unit rates, etc. The project is now refining its initial designs for the key CRNs listed above. These are being simulated and optimized. Experimental demonstrations are being made of each CRN. The project has potentially a transformative impact on research in DNA CRNs due to the speed-ups. The speed-ups also significantly impact many applications of PSD-based CRNs: (a) the amplification CRNs allow speedy nucleic acid detection for diagnostic use in medicine and detection use for forensics, (b) the consensus CRNs allow multiple molecular-device voting, (c) the oscillation CRNs allow for synchronization of multiple repeated molecular-scale operations, and (d) the linear control CRNs allow for regulation of molecular concentrations, for control of a large variety of synthetic and natural biochemical systems. The project is investigating these applications (a-d) and plans to experimentally demonstrate at least one of them.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

细胞和分子过程的调节通常涉及复杂的生化过程，这些过程称为化学反应网络（CRN）。使用DNA分子上的反应的合成CRN可以系统地设计为近似复杂的生化过程。但是，CRN的大多数先前实验方案都依赖于DNA链 - 置换杂交或酶促反应，而所得的合成系统通常会遭受缓慢的速率或泄漏的反应。相比之下，可以通过DNA酶链脱位聚合酶（PSD）获得更高的反应速率。该项目仅使用DNA杂交和PSD反应研究了多种快速和健壮的CRN。该项目包括设计，仿真和实验实现。此外，该项目是高度跨学科的，并影响了本科和研究生级别的跨学科教育。该项目与来自不同学科的不同学术级别的指导和教学学术层面的学生（重点关注女性和代表性不足的少数民族）。 DNA计算和CRN的动手演示是为杜克大学和当地高中的外展计划而设计的。讲习班和讲座正在传播有关基于PSD的高级纳米科学概念的知识，以了解本科和研究生的受众。细胞和分子过程的调节通常涉及复杂的生化过程，这些过程称为化学反应网络（CRN）。使用DNA分子上的反应的合成CRN可以系统地设计为近似复杂的生化过程。但是，CRN的大多数先前实验方案都依赖于DNA链 - 置换杂交或酶促反应，而所得的合成系统通常会遭受缓慢的速率或泄漏的反应。相比之下，DNA酶链脱位聚合酶（PSD）可以获得更高的反应速率。该项目仅使用DNA杂交和PSD反应研究了多种快速和健壮的CRN。该项目包括设计，仿真和实验实现。该项目中研究的关键CRN包括（i）自催化放大器，（ii）动态振荡系统，（iii）分子尺度共识方案，以及（iv）线性控制系统。所有这些CRN都有重要的实际应用。该项目已经完成了使用PSD的一些简单CRN的设计，模拟和初步实验，包括使用PSD进行动态CRN的硅术演示，该psd提供了这些简单CRN的反应速率，泄漏和假阳性的估计。此外，该项目已经完成了使用PSD的动态振荡CRN系统的设计，仿真和实验演示，以确定周期的数量，循环的精度，对初始浓度的敏感性以及对单位速率的近似等等。这些正在模拟和优化。每个CRN都在实验演示。由于加速，该项目可能对DNA CRN的研究产生了变革性的影响。 The speed-ups also significantly impact many applications of PSD-based CRNs: (a) the amplification CRNs allow speedy nucleic acid detection for diagnostic use in medicine and detection use for forensics, (b) the consensus CRNs allow multiple molecular-device voting, (c) the oscillation CRNs allow for synchronization of multiple repeated molecular-scale operations, and (d) the linear control CRNs allow for调节分子浓度，以控制各种合成和自然生化系统。该项目正在调查这些申请（A-D），并计划在实验中至少证明其中一个。该奖项反映了NSF的法定任务，并认为使用基金会的知识分子优点和更广泛的影响审查标准，认为值得通过评估来获得支持。