RNAi-based Boolean gates in the yeast Saccharomyces cerevisiae

Boolean gates, the fundamental components of digital circuits, have been widely investigated in synthetic biology because they permit the fabrication of biosensors and facilitate biocomputing. This study was conducted to design and construct Boolean gates in the yeast Saccharomyces cerevisiae, the main component of which was the RNA interference pathway (RNAi) that is naturally absent from the budding yeast cells. We tested different expression cassettes for the siRNA precursor (a giant hairpin sequence, a DNA fragment-flanked by one or two introns-between convergent promoters or transcribed separately in the sense and antisense directions) and placed different components under the control of the circuit inputs (i.e., the siRNA precursor or proteins such as the Dicer and the Argonaute). We found that RNAi-based logic gates are highly sensitive to promoter leakage and, for this reason, challenging to implement in vivo. Convergent-promoter architecture turned out to be the most reliable solution, even though the overall best performance was achieved with the most difficult design based on the siRNA precursor as a giant hairpin.