Single‐Stranded Interruptions in the T7 Promoter Non‐Template Strand Exhibit Unexpected Transcription Behavior

T7 RNA polymerase (T7RNAP) is widely used for in vitro transcription due to its high specificity and efficiency. Its ability to initiate transcription from a promoter containing single-stranded interruptions, however, remains largely unexplored even though such an ability might provide a potential mechanism for transcriptional regulation in vitro. Here, we systematically introduce single-stranded gaps of varying sizes and positions within the non-template strand of the T7 promoter to investigate their effects on transcription, hypothesizing that small gaps would essentially be tolerated while large gaps would be inhibitory. Surprisingly, single-nucleotide gaps in the promoter's specificity region (-11 to -8) prevented transcription and larger gaps allowed transcription. Fluorescence and gel-based assays confirmed these effects. Structurally, small gaps may disrupt essential hydrogen bonding while larger gaps may increase promoter interaction with T7RNAP, thereby improving binding and initiation while hindering promoter release. These insights reveal new aspects of T7RNAP function and suggest novel strategies for DNA-based transcriptional control in bio-hybrid systems, which we demonstrate by introducing an RNA-dependent transcription-based molecular switch.