Temporal Programming of Cell-Free Transcription Using Orthogonal Enzyme-Responsive DNA Blockers

We report here the design of orthogonal, enzyme-driven DNA transcriptional timers that enable precise programming of time delays in cell-free in vitro transcription. These timers utilize blocker strands that transiently bind to the promoter domain, preventing transcription onset. Selective enzymatic cleavage of the blocker strands triggers their removal, allowing input DNA strands to bind and initiate transcription. By tuning the kinetics of enzymatic blocker degradation─through varying enzyme or blocker strand concentrations─we achieve fine temporal control over transcription half-life (t1/2) from 0.48 ± 0.02 h up to 8.4 ± 0.1 h. Using three different blocker-degrading enzymes (RNase H, uracil-DNA glycosylase (UDG), and formamidopyrimidine DNA glycosylase (Fpg)), we also demonstrate orthogonal temporal control of multiple transcription templates in a single solution. Finally, we show the programmed termination control and downstream regulation of Cas12a enzymatic collateral cleavage activity through such transcription timers. Together, these orthogonal DNA transcriptional timers establish a generalizable and straightforward framework for programming time-resolved transcription and gene expression in cell-free synthetic biology.