(113h) Engineering Self-Contained DNA Circuit for Detection of Proximity-Based Biological Events

Yung, L. Y. L., National University of Singapore
Ang, Y. S., National University of Singapore

Interactions between biomolecules in proximity, such as nucleic acid and proteins, are important for understanding cellular processes and have functional implications in, for example, disease progression. However, a simple method for the in situ detection of such interaction events without the need for complicated enzymatic manipulation or tedious washing steps is lacking in the current molecular toolbox. Herein, we designed a purely DNA circuit capable of autonomously assembling into localized, amplified signal in a single mixing step without further separation step. The circuit composed of three key modules: (1) specific target recognition by an aptamer sequence which induced the opening of a hairpin molecular switch to initiate (2) signal transduction across different binding sites and eventually trigger (3) hybridization chain reaction (HCR) for the development of signal readout in the form of DNA polymerization. The entire circuit was engineered to be self-contained, i.e. it can self-assemble to form localized signal in response to specific binding events on the target molecule. α-thrombin was used as a model protein to evaluate the performance of the individual modules and the overall circuit for proximity interaction under physiologically-relevant buffer condition. The molecular switch opening step was determined to be rate limiting in a kinetics study. The circuit kinetics was modulated through temperature control and equilibrium was achieved within 30 minutes at 37 °C. Good selectivity was achieved in the presence of non-specific protein (e.g. bovine serum albumin) and interfering serum matrix (up to 10% fetal bovine serum). Also, the circuit successfully detected for physiologically-relevant a-thrombin concentration (50 nM – 5 μM). We also demonstrated the feasibility of enhancing the kinetics of localized signal formation at the binding site through the control of temperature and probe concentration. Overall, this work provides a basic general framework from which other circuit modules can be adapted for the in situ detection of other biomolecular or cellular interaction of interest.