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Title: Incorporation of isotopic, fluorescent, and heavy-atom-modified nucleotides into RNAs by position-selective labeling of RNA

Here, site-specific incorporation of labeled nucleotides is an extremely useful synthetic tool for many structural studies (e.g., NMR, electron paramagnetic resonance (EPR), fluorescence resonance energy transfer (FRET), and X-ray crystallography) of RNA. However, specific-position-labeled RNAs >60 nt are not commercially available on a milligram scale. Position-selective labeling of RNA (PLOR) has been applied to prepare large RNAs labeled at desired positions, and all the required reagents are commercially available. Here, we present a step-by-step protocol for the solid-liquid hybrid phase method PLOR to synthesize 71-nt RNA samples with three different modification applications, containing (i) a (CN)-C-13-N-15-labeled segment; (ii) discrete residues modified with Cy3, Cy5, or biotin; or (iii) two iodo-U residues. The flexible procedure enables a wide range of downstream biophysical analyses using precisely localized functionalized nucleotides. All three RNAs were obtained in <2 d, excluding time for preparing reagents and optimizing experimental conditions. With optimization, the protocol can be applied to other RNAs with various labeling schemes, such as ligation of segmentally labeled fragments.
Authors:
 [1] ;  [2] ;  [3] ;  [4] ;  [4] ;  [5] ; ORCiD logo [6] ;  [3] ;  [3]
  1. Shanghai Tong Univ., Shanghai (China); National Institutes of Health, Frederick, MD (United States)
  2. Univ. of Colorado, Boulder, CO (United States); Univ. of Zurich, Zurich (Switzerland)
  3. National Institutes of Health, Frederick, MD (United States)
  4. Argonne National Lab. (ANL), Argonne, IL (United States)
  5. Univ. of Colorado, Boulder, CO (United States)
  6. Univ. of Texas Health Science Center, San Antonio, TX (United States)
Publication Date:
Grant/Contract Number:
AC02-06CH11357
Type:
Accepted Manuscript
Journal Name:
Nature Protocols
Additional Journal Information:
Journal Volume: 13; Journal Issue: 5; Journal ID: ISSN 1754-2189
Publisher:
Nature Publishing Group
Research Org:
Argonne National Lab. (ANL), Argonne, IL (United States)
Sponsoring Org:
National Institutes of Health (NIH), National Cancer Institute; National Science Foundation (NSF); National Institute of Standards and Technology (NIST); USDOE
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES
OSTI Identifier:
1461437

Liu, Yu, Holmstrom, Erik, Yu, Ping, Tan, Kemin, Zuo, Xiaobing, Nesbitt, David J., Sousa, Rui, Stagno, Jason R., and Wang, Yun -Xing. Incorporation of isotopic, fluorescent, and heavy-atom-modified nucleotides into RNAs by position-selective labeling of RNA. United States: N. p., Web. doi:10.1038/nprot.2018.002.
Liu, Yu, Holmstrom, Erik, Yu, Ping, Tan, Kemin, Zuo, Xiaobing, Nesbitt, David J., Sousa, Rui, Stagno, Jason R., & Wang, Yun -Xing. Incorporation of isotopic, fluorescent, and heavy-atom-modified nucleotides into RNAs by position-selective labeling of RNA. United States. doi:10.1038/nprot.2018.002.
Liu, Yu, Holmstrom, Erik, Yu, Ping, Tan, Kemin, Zuo, Xiaobing, Nesbitt, David J., Sousa, Rui, Stagno, Jason R., and Wang, Yun -Xing. 2018. "Incorporation of isotopic, fluorescent, and heavy-atom-modified nucleotides into RNAs by position-selective labeling of RNA". United States. doi:10.1038/nprot.2018.002. https://www.osti.gov/servlets/purl/1461437.
@article{osti_1461437,
title = {Incorporation of isotopic, fluorescent, and heavy-atom-modified nucleotides into RNAs by position-selective labeling of RNA},
author = {Liu, Yu and Holmstrom, Erik and Yu, Ping and Tan, Kemin and Zuo, Xiaobing and Nesbitt, David J. and Sousa, Rui and Stagno, Jason R. and Wang, Yun -Xing},
abstractNote = {Here, site-specific incorporation of labeled nucleotides is an extremely useful synthetic tool for many structural studies (e.g., NMR, electron paramagnetic resonance (EPR), fluorescence resonance energy transfer (FRET), and X-ray crystallography) of RNA. However, specific-position-labeled RNAs >60 nt are not commercially available on a milligram scale. Position-selective labeling of RNA (PLOR) has been applied to prepare large RNAs labeled at desired positions, and all the required reagents are commercially available. Here, we present a step-by-step protocol for the solid-liquid hybrid phase method PLOR to synthesize 71-nt RNA samples with three different modification applications, containing (i) a (CN)-C-13-N-15-labeled segment; (ii) discrete residues modified with Cy3, Cy5, or biotin; or (iii) two iodo-U residues. The flexible procedure enables a wide range of downstream biophysical analyses using precisely localized functionalized nucleotides. All three RNAs were obtained in <2 d, excluding time for preparing reagents and optimizing experimental conditions. With optimization, the protocol can be applied to other RNAs with various labeling schemes, such as ligation of segmentally labeled fragments.},
doi = {10.1038/nprot.2018.002},
journal = {Nature Protocols},
number = 5,
volume = 13,
place = {United States},
year = {2018},
month = {4}
}