UC San Diego

Bacteriophage T7 RNA polymerase (T7 RNAP) is frequently used for RNA synthesis with unnatural base pairs (UBPs) and specific synthetic alterations in a wide range of biotechnological and medicinal applications. As a knowledge gap in the area, however, the molecular basis for recognition and processing of UBPs by T7 RNAP during transcription remains poorly understood. We explored how the hydrophobic Ds–Pa pair is recognized and processed as a third base pair by T7 RNAP during transcription elongation. T7 RNAP integrates DsTP opposite Pa with great efficiency, equivalent to that of natural nucleotides, although the kinetics of PaTP incorporation opposite Ds is significantly slower. Using structural biology approach, we discovered that T7 RNAP recognizes unnatural substrates differently than its natural substrates. We found distinct unnatural nucleoside triphosphate binding sites for PaTP and DsTP at the pre-insertion state. We identified several separate-of-function mutants of T7 RNAP that affect UBP transcription selectively but not normal nucleic acid transcription. These results provide molecular insights into the recognition of UBP transcription by T7 RNAP. Our investigations also provide important information for the creation of the next generation of UBPs for efficient transcription and other applications.