Engineering a robust DNA split proximity circuit with minimized circuit leakage

DNA circuit is a versatile and highly-programmable toolbox which can potentially be used for the autonomous sensing of dynamic events, such as biomolecular interactions. However, the experimental implementation of in silico circuit designs has been hindered by the problem of circuit leakage. Here, we systematically analyzed the sources and characteristics of various types of leakage in a split proximity circuit which was engineered to spatially probe for target sites held within close proximity. Direct evidence that 3'-truncated oligonucleotides were the major impurity contributing to circuit leakage was presented. More importantly, a unique strategy of translocating a single nucleotide between domains, termed 'inter-domain bridging', was introduced to eliminate toehold-independent leakages while enhancing the strand displacement kinetics across a three-way junction. We also analyzed the dynamics of intermediate complexes involved in the circuit computation in order to define the working range of domain lengths for the reporter toehold and association region respectively. The final circuit design was successfully implemented on a model streptavidin-biotin system and demonstrated to be robust against both circuit leakage and biological interferences. We anticipate that this simple signal transduction strategy can be used to probe for diverse biomolecular interactions when used in conjunction with specific target recognition moieties.