The development of unnatural base pairs (UBPs) has greatly increased the information storage capacity of DNA, allowing for transcription of unnatural RNA by the heterologously expressed T7 RNA polymerase (RNAP) in Escherichia coli. However, little is known about how UBPs are transcribed by cellular RNA polymerases. Here, we investigated how synthetic unnatural nucleotides, NaM and TPT3, are recognized by eukaryotic RNA polymerase II (Pol II) and found that Pol II is able to selectively recognize UBPs with high fidelity when dTPT3 is in the template strand and rNaMTP acts as the nucleotide substrate. Our structural analysis and molecular dynamics simulation provide structural insights into transcriptional processing of UBPs in a stepwise manner. Intriguingly, we identified a novel 3′-RNA binding site after rNaM addition, termed the swing state. These results may pave the way for future studies in the design of transcription and translation strategies in higher organisms with expanded genetic codes.

非天然碱基对 (UBP) 的发展极大地增加了 DNA 的信息存储能力，允许通过大肠杆菌中异源表达的 T7 RNA 聚合酶 (RNAP) 转录非天然 RNA. 然而，关于 UBPs 如何被细胞 RNA 聚合酶转录的知之甚少。在这里，我们研究了合成的非天然核苷酸 NaM 和 TPT3 如何被真核 RNA 聚合酶 II (Pol II) 识别，并发现当 dTPT3 在模板链中并且 rNaMTP 充当核苷酸底物。我们的结构分析和分子动力学模拟逐步提供了对 UBP 转录处理的结构见解。有趣的是，我们在添加 rNaM 后发现了一个新的 3'-RNA 结合位点，称为摇摆状态。这些结果可能为未来在具有扩展遗传密码的高等生物中设计转录和翻译策略的研究铺平道路。