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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. © 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.

Citation

Juntaek Oh, Ji Shin, Ilona Christy Unarta, Wei Wang, Aaron W Feldman, Rebekah J Karadeema, Liang Xu, Jun Xu, Jenny Chong, Ramanarayanan Krishnamurthy, Xuhui Huang, Floyd E Romesberg, Dong Wang. Transcriptional processing of an unnatural base pair by eukaryotic RNA polymerase II. Nature chemical biology. 2021 Aug;17(8):906-914

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PMID: 34140682

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