Borderline Boolean states improve the biosensing applications of DNA circuits

Molecular circuits have been used in a wide range of diagnosis applications, from the detection of chemical molecules in solution to the complex processing of cell surface receptors. One of the most important challenges of these systems is the lack of distinguishability between different circuit states when each circuit state represents a specific disease. In this work, we designed a molecular amplification circuit with borderline Boolean states that each state can be distinguished with different color intensity. For this purpose, two DNA complexes and four DNA hairpin structures were designed to detect miR-218 and miR-215 biomarkers. One of the designed DNA complexes has two G-quadruplex structures and the other has only one G-quadruplex structure. In the absence of the inputs, all three G-quadruplex structures are active and produce a high-intensity signal, while in the other three states, including the presence of miR-218, the presence of miR-215, and the presence of both inputs, respectively, one, two, and zero G-quadruplex structures are active. Therefore, the designed system can identify two different biomarkers simultaneously with different signal ratios, which can easily distinguish the different states of the circuit. This strategy is very promising to identify diseases in which any combination of biomarkers leads to a particular disease.