Latent Toehold-Mediated DNA Circuits Based on a Bulge-Loop Structure for Leakage Reduction and Its Application to Signal-Amplifying DNA Logic Gates

DNA circuits based on successive toehold-mediated DNA displacement reactions, particularly entropy-driven DNA circuit (EDC) systems, have attracted considerable attention as powerful enzyme-free tools for dynamic DNA nanotechnology. However, background leakage (noise signal) often occurs when the circuit is executed nonspecifically, even in the absence of the appropriate catalyst DNA (input). This study designed and developed a new latent toehold-mediated DNA circuit (LDC) system that relies on a bulge-loop structure as a latent toehold toward leakage reduction. Furthermore, the number (size) of nucleotides (nt) in the bulge-loop is found to play a significant role in the performance (i.e., leakage, signal, and kinetics) of LDC systems. In fact, the signal rate for the LDC systems increased as the number of nt in the bulge-loop increased from 4 to 8, whereas the leakage rate of the LDC systems with bulge-loops of 7 nt or less was low, but the leakage rate of the LDC system with a bulge-loop of 8 nt increased significantly. Note that the LDC system with the optimal bulge-loop (7 nt) was capable of not only reducing the leakage but also accelerating the circuit speed without any signal loss, unlike methods of reducing the leakage by reducing the signal reported previously for the conventional EDC systems. These facts indicate that the 7 nt bulge-loop acts as a "latent" toehold for the DNA circuit system. By using the amplification function of output signals with an accelerated circuit and reduced leakage, our LDC system with a 7 nt bulge-loop could be applied directly and successfully to signal-amplifying DNA logic gates such as OR and AND gates, and thus, sufficient output signals could be obtained even with a small amount of input. These findings reveal that our LDC systems with a bulge-loop structure can replace the conventional EDC system and have enormous potential in the field of DNA nanotechnology.