Alternate Strategies to Induce Dynamically Modulated Transient Transcription Machineries

Emulating native transient transcription machineries modulating temporal gene expression by synthetic circuits is a major challenge in the area of systems chemistry. Three different methods to operate transient transcription machineries and to modulate the gated transcription processes of target RNAs are introduced. One method involves the design of a reaction module consisting of transcription templates being triggered by promoter fuel strands transcribing target RNAs and in parallel generating functional DNAzymes in the transcription templates, modulating the dissipative depletion of the active templates and the transient operation of transcription circuits. The second approach involves the application of a reaction module consisting of two transcription templates being activated by a common fuel promoter strand. While one transcription template triggers the transcription of the target RNA, the second transcription template transcribes the anti-fuel strand, displacing the promoter strand associated with the transcription templates, leading to the depletion of the transcription templates and to the dynamic transient modulation of the transcription process. The third strategy involves the assembly of a reaction module consisting of a reaction template triggered by a fuel promoter strand transcribing the target RNA. The concomitant nickase-stimulated depletion of the promoter strand guides the transient modulation of the transcription process. Via integration of two parallel fuel-triggered transcription templates in the three transcription reaction modules and application of template-specific blocker units, the parallel and gated transiently modulated transcription of two different RNA aptamers is demonstrated. The nickase-stimulated transiently modulated transcription reaction module is applied as a functional circuit guiding the dynamic expression of gated, transiently operating, catalytic DNAzymes.