Division of Pharmaceutical Sciences, Skaggs School of Pharmacy & Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA, USA.
Institute of Bioengineering and Bioimaging (IBB), Agency for Science, Technology and Research (A*STAR), Singapore, Singapore.
Nat Commun. 2023 Jan 13;14(1):195. doi: 10.1038/s41467-022-35755-8.
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.
T7 RNA 聚合酶（T7 RNAP）被广泛用于合成具有合成修饰和非天然碱基对（UBP）的 RNA 分子，用于各种生物技术和治疗应用。然而，T7 RNAP 对 UBP 的转录识别的分子基础仍知之甚少。在这里，我们专注于一个代表性的 UBP，即 7-(2-噻吩基）-咪唑[4,5-b]吡啶（Ds）和吡咯-2-甲醛（Pa），并研究了疏水 Ds-Pa 对如何被 T7 RNAP 识别。我们的动力学分析表明，T7 RNAP 选择性地识别模板中的 Ds 或 Pa 碱基，并优先将其对应的非天然碱基核苷酸底物（PaTP 或 DsTP）掺入到转录中，而不是天然 NTPs。我们的结构研究表明，与天然底物相比，T7 RNAP 在预插入状态以独特的方式识别非天然底物。这些结果为 T7 RNAP 对 UBP 的转录识别提供了机制见解，并为设计下一代 UBP 提供了有价值的信息。
