RNAi-based Boolean gates in the yeast Saccharomyces cerevisiae

Area of Science:

• Synthetic Biology
• Molecular Biology
• Biocomputing

Background:

• Boolean gates are foundational to digital circuits and are increasingly explored in synthetic biology for biosensors and biocomputing.
• The RNA interference (RNAi) pathway, crucial for gene silencing, is naturally absent in yeast Saccharomyces cerevisiae, presenting an opportunity for synthetic implementation.

Purpose of the Study:

• To design and construct functional Boolean logic gates within yeast using the RNA interference pathway.
• To evaluate different genetic circuit architectures and components for optimal RNAi-based gate performance in vivo.

Main Methods:

• Investigated various expression cassettes for small interfering RNA (siRNA) precursors, including hairpin sequences and intron-flanked DNA fragments.
• Controlled circuit components (siRNA precursor, Dicer, Argonaute) using different promoter strategies and circuit inputs.
• Assessed the performance and reliability of RNAi-based logic gates in Saccharomyces cerevisiae, focusing on challenges like promoter leakage.

Main Results:

• RNAi-based logic gates are sensitive to promoter leakage, complicating in vivo implementation.
• A convergent-promoter architecture demonstrated the highest reliability among the tested designs.
• The most complex design, utilizing a giant hairpin siRNA precursor, yielded the best overall performance.

Conclusions:

• Implementing RNAi-based Boolean gates in yeast is feasible but requires careful design to mitigate challenges like promoter leakage.
• Convergent-promoter designs offer a robust framework for building biological logic gates.
• Further optimization of siRNA precursor designs can enhance the performance of synthetic RNAi circuits for biocomputing.