Sprecher
Beschreibung
Here, we develop orthogonal enzyme-driven DNA transcriptional timers capable of rationally programming a tunable delay of in vitro transcription onset. These timers are based on blocker strands that, by binding to the promoter region of the DNA template, prevent transcription initiation. The blockers can subsequently be removed via specific enzymatic reactions. Once the blocker strand is removed, an input strand binds and completes the promoter, thus activating transcription. The kinetics of enzymatic blocker degradation control the timing of transcription onset. We designed three tunable timers using RNase H, Uracil-DNA Glycosylase (UDG), and DNA-formamidopyrimidine glycosylase (Fpg) with their respective blocker strands. By varying the concentrations of the blocker or the enzyme, we can modulate the onset of transcription. Moreover, following the same design strategy, we successfully programmed not only the transcriptional activation (turning on) but also the deactivation (turning off) of the reaction. This provides a versatile strategy for rationally programming both the onset and termination of in vitro transcription. Additionally, the orthogonality and high programmability of these timers allow us to control the timing of all three systems in a one-tube reaction. This technology can further be applied to modulate the collateral activity of gene-editing enzymes like Cas12.
