Nucleic acid circuit and its application in genetic diagnostic
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DNA can execute programmed strand exchange reactions that process signals and information. In particular, toehold-mediated strand exchange, a process in which one strand of a hemi-duplex is replaced by another, single strand to create a more stable complex, is the basis for many DNA circuits. By engineering several strand exchange reactions in a systematic way, complex DNA circuits can be created that accomplish sophisticated control tasks, similar to an electronic circuit. In this dissertation, we will demonstrate how we engineered and improved a toehold-mediated strand exchanged-based reaction, catalytic hairpin assembly (CHA), to make it into a real-world nucleic acid diagnostic. While CHA has previously been shown to act as an excellent amplifier of nucleic acid signals, it can sometimes execute non-specifically even in the absence of catalyst, limiting signal-to-noise and limits of detection. By introducing two mismatched bases into a specific domain on the circuit, the background leakage can be greatly decreased and the signal-to-noise ratio can be improved from less than 10 to over 100. However, the improvement of the signal:background ratio still cannot increase sensitivity compatible to the enzyme-based nucleic acid amplification. Still, DNA circuits can improve upon background issues inherent in isothermal amplification reactions, which often produce spurious side products. We engineered DNA circuits that were thermostable from 37 °C to 60 °C and used these for the real-time detection of isothermal amplification reactions. These circuits in essence acted like an additional probe to measure the accumulation of correct, rather than spurious, amplicons. One isothermal amplification reaction, loop-mediated isothermal amplification (LAMP) combined with our DNA could detect particular alleles of M. tuberculosis RNA polymerase (rpoB) in sputum and of the melanoma-related biomarker BRAF. As one more step towards generating a true point-of-care (POC) test, we engineered DNA circuits to transduce amplicons into an off-the-shelf glucometer. Using these reactions and devices we could directly transduce Middle-East respiratory syndrome coronavirus (MERS) and Zaire Ebolavirus (Ebola) templates into glucose signals, with a sensitivity as low as 20-100 copies/µL. Virus from cell lysates and synthetic templates could be readily amplified and detected even in sputum or saliva. An OR gate that coordinately triggered on viral amplicons further guaranteed fail-safe virus detection.