Abstract
Artificially designed molecular systems with programmable behaviors have become a valuable tool in chemistry, biology, material science, and medicine. Although information processing in biological regulatory pathways is remarkably robust to error, it remains a challenge to design molecular systems that are similarly robust. With functionality determined entirely by secondary structure of DNA, strand displacement has emerged as a uniquely versatile building block for cell-free biochemical networks. Here, we experimentally investigate a design principle to reduce undesired triggering in the absence of input (leak), a side reaction that critically reduces sensitivity and disrupts the behavior of strand displacement cascades. Inspired by error correction methods exploiting redundancy in electrical engineering, we ensure a higher-energy penalty to leak via logical redundancy. Our design strategy is, in principle, capable of reducing leak to arbitrarily low levels, and we experimentally test two levels of leak reduction for a core "translator" component that converts a signal of one sequence into that of another. We show that the leak was not measurable in the high-redundancy scheme, even for concentrations that are up to 100 times larger than typical. Beyond a single translator, we constructed a fast and low-leak translator cascade of nine strand displacement steps and a logic OR gate circuit consisting of 10 translators, showing that our design principle can be used to effectively reduce leak in more complex chemical systems.
Highlights
Designed molecular systems with programmable behaviors have become a valuable tool in chemistry, biology, material science, and medicine
Since the translators have the property that all domains have well-defined neighboring domains, the sequence space can be represented by one contiguous sequence (i.e., x1x2y1y2 for the single-long domain (SLD) and double-long domain (DLD) designs)
We further confirmed the theoretical prediction that the total amount of leak in a single-translator plus reporter system at thermodynamic equilibrium is lower in the DLD or the triple-long domain (TLD) schemes compared with the SLD scheme (Fig. 7)
Summary
In contrast to clamping techniques that introduce an energy barrier to overall thermodynamically favorable reactions, forming such large complexes here incurs a thermodynamic (entropic) penalty, making leak unfavorable In principle, this method could offer a systematic way to reduce leak to an arbitrarily low level by increasing N and reducing concentration. We develop a kinetic model that quantitatively captures the leak dynamics at high concentrations, including two distinct timescales of leak that we observed To investigate this leak reduction method, we start with the simplest nontrivial strand displacement cascade, a “translator,” which converts an input into an output of independent sequence. At the high concentrations permitted by the “leakless” design, the desired strand displacement reaction in the presence of input is very fast: the output signal reaches > 80% completion within 3 min of addition of input, while by increasing redundancy, leak could be reduced to the limit of detection in our experimental setting. The fast (order of minutes) and lowleak (less than 5% over 10 h) performance of these circuits shows that leak reduction generalizes to more complex settings
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