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

Abstract

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:
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
OSTI Identifier:
1461437
Grant/Contract Number:  
AC02-06CH11357
Resource 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
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

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., 2018. 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. Thu . "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}
}

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Works referenced in this record:

Structural Basis for Discriminative Regulation of Gene Expression by Adenine- and Guanine-Sensing mRNAs
journal, December 2004


The 3' proximal translational enhancer of Turnip crinkle virus binds to 60S ribosomal subunits
journal, September 2008

  • Stupina, V. A.; Meskauskas, A.; McCormack, J. C.
  • RNA, Vol. 14, Issue 11
  • DOI: 10.1261/rna.1227808

A brief review of DNA and RNA chemical synthesis
journal, March 2011

  • Caruthers, Marvin H.
  • Biochemical Society Transactions, Vol. 39, Issue 2
  • DOI: 10.1042/BST0390575

Isotope labeling strategies for NMR studies of RNA
journal, September 2009

  • Lu, Kun; Miyazaki, Yasuyuki; Summers, Michael F.
  • Journal of Biomolecular NMR, Vol. 46, Issue 1
  • DOI: 10.1007/s10858-009-9375-2

Aptamers: molecular tools for analytical applications
journal, June 2007

  • Mairal, Teresa; Cengiz Özalp, Veli; Lozano Sánchez, Pablo
  • Analytical and Bioanalytical Chemistry, Vol. 390, Issue 4
  • DOI: 10.1007/s00216-007-1346-4

Multiple segmental and selective isotope labeling of large RNA for NMR structural studies
journal, June 2008

  • Nelissen, F. H. T.; van Gammeren, A. J.; Tessari, M.
  • Nucleic Acids Research, Vol. 36, Issue 14
  • DOI: 10.1093/nar/gkn397

Three-state mechanism couples ligand and temperature sensing in riboswitches
journal, July 2013

  • Reining, Anke; Nozinovic, Senada; Schlepckow, Kai
  • Nature, Vol. 499, Issue 7458
  • DOI: 10.1038/nature12378

Nucleic acid aptamers for clinical diagnosis: cell detection and molecular imaging
journal, December 2010

  • Soontornworajit, Boonchoy; Wang, Yong
  • Analytical and Bioanalytical Chemistry, Vol. 399, Issue 4
  • DOI: 10.1007/s00216-010-4559-x

Fluorescent mRNA labeling through cytoplasmic FISH
journal, November 2013

  • Gasnier, Maxime; Dennis, Cynthia; Vaurs-Barrière, Catherine
  • Nature Protocols, Vol. 8, Issue 12
  • DOI: 10.1038/nprot.2013.160

Site-specific fluorescent probing of RNA molecules by unnatural base-pair transcription for local structural conformation analysis
journal, June 2010

  • Hikida, Yasushi; Kimoto, Michiko; Yokoyama, Shigeyuki
  • Nature Protocols, Vol. 5, Issue 7
  • DOI: 10.1038/nprot.2010.77

The Chemical Synthesis of DNA/RNA: Our Gift to Science
journal, December 2012


Dramatic Improvement of Crystals of Large RNAs by Cation Replacement and Dehydration
journal, September 2014


Effects of Mg 2+ on the Free Energy Landscape for Folding a Purine Riboswitch RNA
journal, April 2011

  • Leipply, Desirae; Draper, David E.
  • Biochemistry, Vol. 50, Issue 14
  • DOI: 10.1021/bi101948k

Real-time observation of the transition from transcription initiation to elongation of the RNA polymerase
journal, December 2009

  • Tang, Guo-Qing; Roy, Rahul; Bandwar, Rajiv P.
  • Proceedings of the National Academy of Sciences, Vol. 106, Issue 52
  • DOI: 10.1073/pnas.0906979106

Solution structure of the cap-independent translational enhancer and ribosome-binding element in the 3' UTR of turnip crinkle virus
journal, January 2010

  • Zuo, X.; Wang, J.; Yu, P.
  • Proceedings of the National Academy of Sciences, Vol. 107, Issue 4
  • DOI: 10.1073/pnas.0908140107

Rapid purification of high-activity Taq DNA polymerase
journal, January 1993


Genetic engineering of streptavidin, a versatile affinity tag
journal, September 1998

  • Sano, Takeshi; Vajda, Sandor; Cantor, Charles R.
  • Journal of Chromatography B: Biomedical Sciences and Applications, Vol. 715, Issue 1
  • DOI: 10.1016/S0378-4347(98)00316-8

Rapid Mutagenesis and Purification of Phage RNA Polymerases
journal, February 1997

  • He, Biao; Rong, Mingqing; Lyakhov, Dmitry
  • Protein Expression and Purification, Vol. 9, Issue 1
  • DOI: 10.1006/prep.1996.0663

Experimental phasing with SHELXC / D / E : combining chain tracing with density modification
journal, March 2010

  • Sheldrick, George M.
  • Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 4
  • DOI: 10.1107/S0907444909038360

Single Molecule Fluorescence Approaches Shed Light on Intracellular RNAs
journal, January 2014

  • Pitchiaya, Sethuramasundaram; Heinicke, Laurie A.; Custer, Thomas C.
  • Chemical Reviews, Vol. 114, Issue 6
  • DOI: 10.1021/cr400496q

Optimizing splinted ligation of highly structured small RNAs
journal, December 2005


Synthesis and applications of RNAs with position-selective labelling and mosaic composition
journal, May 2015

  • Liu, Yu; Holmstrom, Erik; Zhang, Jinwei
  • Nature, Vol. 522, Issue 7556
  • DOI: 10.1038/nature14352

In Vitro Transcription: Preparative RNA Yields in Analytical Scale Reactions
journal, August 1994


Major Conformational Changes During T7RNAP Transcription Initiation Coincide with, and are Required for, Promoter Release
journal, October 2005

  • Guo, Qing; Nayak, Dhananjaya; Brieba, Luis G.
  • Journal of Molecular Biology, Vol. 353, Issue 2
  • DOI: 10.1016/j.jmb.2005.08.016

Synthesis, Oxidation Behavior, Crystallization and Structure of 2‘-Methylseleno Guanosine Containing RNAs
journal, August 2006

  • Moroder, Holger; Kreutz, Christoph; Lang, Kathrin
  • Journal of the American Chemical Society, Vol. 128, Issue 30
  • DOI: 10.1021/ja0621400

Touchdown PCR for increased specificity and sensitivity in PCR amplification
journal, August 2008


RNA labeling, conjugation and ligation
journal, June 2011


Specific labeling: An effective tool to explore the RNA world
journal, December 2015


Nucleic acid X-ray crystallography via direct selenium derivatization
journal, January 2011

  • Lin, Lina; Sheng, Jia; Huang, Zhen
  • Chemical Society Reviews, Vol. 40, Issue 9
  • DOI: 10.1039/c1cs15020k

Rapid global structure determination of large RNA and RNA complexes using NMR and small-angle X-ray scattering
journal, October 2010


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