Phosphorothioate-Based Site-Specific Labeling of Large RNAs for Structural and Dynamic Studies

Hu, Yanping; Wang, Yan; Singh, Jaideep; Sun, Ruirui; Xu, Lilei; Niu, Xiaolin; Huang, Keyun; Bai, Guangcan; Liu, Guoquan; Zuo, Xiaobing; Chen, Chunlai; Qin, Peter Z.; Fang, Xianyang

doi:10.1021/acschembio.2c00199

Phosphorothioate-Based Site-Specific Labeling of Large RNAs for Structural and Dynamic Studies

Journal Article · Wed Sep 07 00:00:00 EDT 2022 · ACS Chemical Biology

DOI:https://doi.org/10.1021/acschembio.2c00199· OSTI ID:1970720

Hu, Yanping ^[1]; ^[1]; ^[2]; Sun, Ruirui ^[1]; Xu, Lilei ^[1]; Niu, Xiaolin ^[1]; Huang, Keyun ^[1]; Bai, Guangcan ^[3]; ^[3]; Zuo, Xiaobing ^[4]; ^[1]; ^[2]; ^[1]

Tsinghua University, Beijing (China)
University of Southern California, Los Angeles, CA (United States)
Peking University, Beijing (China)
Argonne National Laoratory. (ANL), Lemont, IL (United States)

Pulsed electron-electron double resonance (PELDOR) spectroscopy, X-ray scattering interferometry (XSI), and single-molecule Forster resonance energy transfer (smFRET) are molecular rulers that provide inter- or intramolecular pair-wise distance distributions in the nanometer range, thus being ideally suitable for structural and dynamic studies of biomolecules including RNAs. The prerequisite for such applications requires site-specific labeling of biomolecules with spin labels, gold nanoparticles, and fluorescent tags, respectively. Recently, site-specific labeling of large RNAs has been achieved by a combination of transcription of an expanded genetic alphabet containing A-T/G-C base pairs and NaM-TPT3 unnatural base pair (UBP) with posttranscriptional modifications at UBP bases by click chemistry or amine-NHS ester reactions. However, due to the bulky sizes of functional groups or labeling probes used, such strategies might cause structural perturbation and decrease the accuracy of distance measurements. Here, we synthesize an a-thiophosphorylated variant of rTPT3TP (rTPT3aS), which allows for post-transcriptional site-specific labeling of large RNAs at the internal a-phosphate backbone via maleimide-modified probes. Subsequent PELDOR, XSI, and smFRET measurements result in narrower distance distributions than labeling at the TPT3 base. The presented strategy provides a new route to empower the molecular rulers for structural and dynamic studies of large RNA and its complex.

View Accepted Manuscript (DOE)

Research Organization:: Argonne National Laboratory (ANL), Lemont, IL (United States)

Sponsoring Organization:: Anton B. Burg Foundation; National Institute of Health (NIH); National Key Research and Development Program of China; National Natural Science Foundation of China (NSFC); National Science Foundation (NSF); USDOE Office of Science (SC)

Grant/Contract Number:: AC02-06CH11357

OSTI ID:: 1970720

Journal Information:: ACS Chemical Biology, Journal Name: ACS Chemical Biology Journal Issue: 9 Vol. 17; ISSN 1554-8929

Publisher:: American Chemical Society (ACS)Copyright Statement

Country of Publication:: United States

Language:: English

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