Characterization of Cascaded DNA Generation Reaction for Amplifying DNA Signal

Abstract

Toward the construction of robotic and cybernetic systems with molecular reactions, development of a reaction that can rapidly generate single-stranded DNA (ssDNA) molecules in response to an input signal is an essential demand. This study explored the cascading of DNA generation reactions employing DNA polymerase and nicking endonuclease to achieve significant amplification of ssDNA molecules serving as signals to direct and fuel the operation of DNA-based systems. The modular architecture allows for interconnection with other reactions through primer-binding sequence or template design, making it adaptable to various molecular robotic components. The research aims to contribute to the development of efficient and reliable amplification circuits for molecular robotics and cybernetics. The cascading reactions, implemented up to three layers, exhibit enhanced amplification rates and sensitivities at physiological temperatures, enabling stable hybridization with complementary sequences. The investigation reveals the potential of the proposed approach to bridge the molecular quantity gap and restore signals in molecular systems including molecular robots and related applications. The experimental validation demonstrates the feasibility of achieving up to 100,000-fold amplification in response to low concentrations of primers within two hours, driving the structural transformation of DNA probes and nanomotors, while suppressing non-specific leak amplification, thereby showcasing practical applicability. The study's findings address fundamental challenges in ssDNA amplification and opens avenues for creating intelligent systems composed of molecular components with increased sensitivity and responsiveness.