DNA Flap‐Mediated Control of Transcription for Programmable RNA Synthesis

ABSTRACT The growing success of RNA‐based therapeutics has emphasized the need for precise and programmable RNA synthesis platforms. T7 RNA polymerase (T7RP) is widely utilized for in vitro transcription; however, most existing regulatory strategies rely on auxiliary proteins or chemical modulators. Here, we investigated whether transcription can be regulated solely through nucleic acid sequences. Specifically, we evaluated the effects of single‐stranded DNA flap sequences appended to the 3′ end of the non‐template strand of the T7 promoter, termed the flap promoter, on transcriptional efficiency. Remarkably, we observed a sequence‐dependent inhibitory effect, wherein flaps enriched in pyrimidines (cytosine and thymine) significantly suppressed T7RP‐mediated transcription. Leveraging this intrinsic sequence preference, we developed two novel transcription control platforms, D‐FIT (DNAzyme‐mediated Flap promoter Induced Transcription control) and M‐FIT (MNAzyme‐mediated Flap promoter Induced Transcription control) that enable precise regulation of T7RP activity without the need for auxiliary proteins or chemical agents. These findings uncover a previously unrecognized sequence‐specific regulatory mechanism of T7RP and establish a new framework for the rational design of programmable RNA synthesis systems, with broad potential applications in RNA therapeutics and diagnostics.