Structural basis of transcription recognition of a hydrophobic unnatural base pair by T7 RNA polymerase

Juntaek Oh, Michiko Kimoto, Haoqing Xu, Jenny Chong, Ichiro Hirao () and Dong Wang ()
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Juntaek Oh: University of California, San Diego
Michiko Kimoto: Agency for Science, Technology and Research (A*STAR)
Haoqing Xu: University of California, San Diego
Jenny Chong: University of California, San Diego
Ichiro Hirao: Agency for Science, Technology and Research (A*STAR)
Dong Wang: University of California, San Diego

Nature Communications, 2023, vol. 14, issue 1, 1-8

Abstract: Abstract Bacteriophage T7 RNA polymerase (T7 RNAP) is widely used for synthesizing RNA molecules with synthetic modifications and unnatural base pairs (UBPs) for a variety of biotechnical and therapeutic applications. However, the molecular basis of transcription recognition of UBPs by T7 RNAP remains poorly understood. Here we focused on a representative UBP, 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds) and pyrrole 2-carbaldehyde (Pa), and investigated how the hydrophobic Ds–Pa pair is recognized by T7 RNAP. Our kinetic assays revealed that T7 RNAP selectively recognizes the Ds or Pa base in the templates and preferentially incorporates their cognate unnatural base nucleotide substrate (PaTP or DsTP) over natural NTPs. Our structural studies reveal that T7 RNAP recognizes the unnatural substrates at the pre-insertion state in a distinct manner compared to natural substrates. These results provide mechanistic insights into transcription recognition of UBP by T7 RNAP and provide valuable information for designing the next generation of UBPs.

Date: 2023
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DOI: 10.1038/s41467-022-35755-8

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