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Posttranscriptional Site-Directed Spin Labeling of Large RNAs with an Unnatural Base Pair System Under Non-Denaturing Conditions

preprint
submitted on 01.04.2020, 14:24 and posted on 06.04.2020, 07:34 by Yan Wang, Venkatesan Kathiresan, Yaoyi Chen, Yanping Hu, Wei Jiang, Guangcan Bai, Guoquan Liu, Peter Z. Qin, Xianyang Fang

Site-directed spin labeling (SDSL) of large RNAs for electron paramagnetic resonance (EPR) spectroscopy remains challenging up-to-date. We here demonstrate an efficient and generally applicable posttranscriptional SDSL method for large RNAs under non-denaturing conditions using an expanded genetic alphabet containing the NaM-TPT3 unnatural base pair (UBP). An alkyne-modified TPT3 ribonucleotide triphosphate (rTPT3COTP) is synthesized and site-specifically incorporated into large RNAs by in vitro transcription, which allows attachment of the azide-containing nitroxide through click chemistry. We validate this strategy using a 419-nucleotide Ribonuclease P (RNase P) RNA from Bacillus stearothermophilus. The effects of site-directed UBP incorporation and subsequent spin labeling to global structure and function of RNase P are marginal as evaluated by Circular Dichroism spectroscopy, Small Angle X-ray Scattering, and enzymatic assay. Continuous-wave EPR analyses reveal that the labeling reaction is efficient and specific, and Pulsed Electron-Electron Double Resonance measurements yield an inter-spin distance distribution that agrees well with the crystal structure. Thus, the labeling strategy as presented overcomes the size constraint of RNA labeling, opening new possibilities for application of EPR spectroscopy in investigating structure and dynamics of large RNA.


Funding

Xianyang Fang, NNSF No.U1832215

Peter.Z.Qin,MCB-1818107

History

Email Address of Submitting Author

wangyan16@mails.tsinghua.edu.cn

Institution

Tsinghua University

Country

China

ORCID For Submitting Author

0000-0002-0452-6750

Declaration of Conflict of Interest

There are no conflicts of interest to declare.

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