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

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.