DNA-based circuits and computational tools offer great potential for advanced biomedical and technological applications. However, leakage, which is the production of an output in the absence of an input, widely exists in DNA network. As a new approach to leakage reduction, this study utilizes availability to reduce leakage in an entropy-driven autocatalytic DNA reaction networks. Here, we report the performance improvements resulting from direct tailoring of fuel strand availability through two novel approaches: (1) the addition of interfering domains to fuel strands, and (2) the introduction of separate small interfering strands. The best performing fuel designs resulted in increased performance ratios of up to 22%. Employing small interfering strands (5−12 nucleotides (nt)) improved the performance ratios by up to 21%. Furthermore, the stability of the network using either leakage reduction method matched well with computed availability and experimental results showing Spearman correlation coefficients of −0.84 for modified fuel strands and −0.92 for small interfering strands.

DNA strand displacement networks are a critical part of dynamic DNA nanotechnology and are proven primitives for implementing chemical reaction networks. Precise kinetic control of these networks is important for their use in a range of applications. Among the better understood and widely leveraged kinetic properties of these networks are toehold sequence, length, composition, and location. While steric hindrance has been recognized as an important factor in such systems, a clear understanding of its impact and role is lacking. Here, a systematic investigation of steric hindrance within a DNA toehold-mediated strand displacement network was performed through tracking kinetic reactions of reporter complexes with incremental concatenation of steric moieties near the toehold. Two subsets of steric moieties were tested with systematic variation of structures and reaction conditions to isolate sterics from electrostatics. Thermodynamic and coarse-grained computational modeling was performed to gain further insight into the impacts of steric hindrance. Steric factors yielded up to 3 orders of magnitude decrease in the reaction rate constant. This pronounced effect demonstrates that steric moieties can be a powerful tool for kinetic control in strand displacement networks while also being more broadly informative of DNA structural assembly in both DNA-based therapeutic and diagnostic applications that possess elements of steric hindrance through DNA functionalization with an assortment of chemistries.

Solution-based biosensors that utilize aptamers have been engineered in a variety of formats to detect a range of analytes for both medical and environmental applications. However, since aptamers have fixed base sequences, incorporation of aptamers into DNA strand displacement networks for feed-forward signal amplification and processing requires significant redesign of downstream DNA reaction networks. We designed a novel aptamer transduction network that releases customizable output domains, which can then be used to initiate downstream strand displacement reaction networks without any sequence redesign of the downstream reaction networks. In our aptamer transducer (AT), aptamer input domains are independent of output domains within the same DNA complex and are reacted with a fuel strand after aptamer–ligand binding. ATs were designed to react with two fluorescent dye-labeled reporter complexes to show the customizability of the output domains, as well as being used as feed-forward inputs to two previously studied catalytic reaction networks, which can be used as amplifiers. Through our study, we show both successful customizability and feed-forward capability of our ATs.