Rsearchers use such a transcriptional oscillator as a molecular clock to time two other molecular processes. One of these is a DNA nanomechanical device, a DNA tweezers that gives different fluorescent signals when open and when closed. Different RNA molecules produced by the transcriptional oscillator act to either open or close the tweezers. The other process is the synthesis by the transcriptional oscillator of an RNA molecule that binds the dye molecule Malachite Green, changing its fluorescence.
The interplay between different genes can give rise to periodic, oscillatory expression patterns, and some preliminary experiments have demonstrated the potential to design synthetic systems that recapitulate such behavior and act as molecular ‘timers.’ By building on previous work with nucleic acid-based oscillators, Franco et al. have obtained valuable insights that might lead to improved stability for such constructs.
To begin, they performed numerical simulations to examine the oscillatory behavior of a system driven by the in vitro transcription of two small ‘genelets,’ each of which produces an RNA molecule that regulates the activity of the other.
After identifying stable operating conditions, they introduced a ‘load’—a molecule that generates detectable output in response to oscillator activity. The researchers noted that the core oscillator tended to be destabilized when it was coupled directly to load activation. However, the introduction of an insulator ‘genelet,’ which gets switched on by oscillator output RNAs and in turn produces a secondary signal that interacts with the load, enabled the system to function stably even in the presence of large and potentially disruptive load concentrations
The construction of synthetic biochemical circuits from simple components illuminates how complex behaviors can arise in chemistry and builds a foundation for future biological technologies. A simplified analog of genetic regulatory networks, in vitro transcriptional circuits, provides a modular platform for the systematic construction of arbitrary circuits and requires only two essential enzymes, bacteriophage T7 RNA polymerase and Escherichia coli ribonuclease H, to produce and degrade RNA signals. In this study, we design and experimentally demonstrate three transcriptional oscillators in vitro. First, a negative feedback oscillator comprising two switches, regulated by excitatory and inhibitory RNA signals, showed up to five complete cycles. To demonstrate modularity and to explore the design space further, a positive-feedback loop was added that modulates and extends the oscillatory regime. Finally, a three-switch ring oscillator was constructed and analyzed. Mathematical modeling guided the design process, identified experimental conditions likely to yield oscillations, and explained the system’s robust response to interference by short degradation products. Synthetic transcriptional oscillators could prove valuable for systematic exploration of biochemical circuit design principles and for controlling nanoscale devices and orchestrating processes within artificial cells.