(335f) Engineering a Robust DNA Split Proximity Circuit for Probing Proximal Biological Recognition Events

Authors: 
Yung, L. Y. L., National University of Singapore
DNA circuit is a modular and highly-programmable toolbox which can be designed for the autonomous probing of dynamic events, e.g. biomolecular interactions. In this talk, we will introduce a â??plug-and-playâ? DNA split proximity circuit which was engineered to spatially probe for recognition events occurring within close proximity. The central computing module, driven by the concept of association toehold, served as a signal transducer to convert diverse recognition events (e.g. DNA hybridization, aptamer-thrombin binding, small molecule-protein interaction) into readout signals (e.g. fluorophore-quencher and hybridization chain reaction).

We recognize that a major common problem hindering the successful implementation of several in silico circuit designs is the presence of undesired circuit leakage. To ensure the robustness and practical relevance of our proposed design, we focused on identifying the sources and characterizing various types of leakage in order to devise effective counter strategies. Three key findings which are generally applicable to other DNA circuits will be shared in this presentation.

First, we provided direct experimental evidence that 3â??-truncated oligonucleotides as the major defected product in solid-state synthesis was the main culprit for initial leakage. Next, we introduced a unique strategy, termed â??inter-domain bridgingâ?, by translocating a single nucleotide across split domains to eliminate toehold-independent hybridization while improving the strand displacement kinetics across the three-way junction by ca. 13-fold and enhancing the equilibrium signal by one-fold. Finally, we found that asymptotic leakage was inevitable given the inherent dynamics of the intermediate complexes involved in a three-way junction circuit. The best counter-strategy was to use the shortest possible association length, which we recommend to be less than 6 nt.

These three key design guidelines culminated in a final optimized circuit design which was successfully implemented on a model streptavidin-biotin system. The circuit operated robustly against both circuit leakages and interferences from non-specific proteins or a biological environment of 10% fetal bovine serum. A limit of detection of 2.95 nM was achieved within 10 min of detection. 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. Successful systems tested in our lab include thrombin-aptamer binding, HER2 homodimerization and DNA hybridization.