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:
-
[6];
- Shanghai Tong Univ., Shanghai (China); National Institutes of Health, Frederick, MD (United States)
- Univ. of Colorado, Boulder, CO (United States); Univ. of Zurich, Zurich (Switzerland)
- National Institutes of Health, Frederick, MD (United States)
- Argonne National Lab. (ANL), Argonne, IL (United States)
- Univ. of Colorado, Boulder, CO (United States)
- 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. https://doi.org/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. https://doi.org/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
- Serganov, Alexander; Yuan, Yu-Ren; Pikovskaya, Olga
- Chemistry & Biology, Vol. 11, Issue 12
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
A brief review of DNA and RNA chemical synthesis
journal, March 2011
- Caruthers, Marvin H.
- Biochemical Society Transactions, Vol. 39, Issue 2
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
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
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
Three-state mechanism couples ligand and temperature sensing in riboswitches
journal, July 2013
- Reining, Anke; Nozinovic, Senada; Schlepckow, Kai
- Nature, Vol. 499, Issue 7458
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
Fluorescent mRNA labeling through cytoplasmic FISH
journal, November 2013
- Gasnier, Maxime; Dennis, Cynthia; Vaurs-Barrière, Catherine
- Nature Protocols, Vol. 8, Issue 12
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
The Chemical Synthesis of DNA/RNA: Our Gift to Science
journal, December 2012
- Caruthers, Marvin H.
- Journal of Biological Chemistry, Vol. 288, Issue 2
Dramatic Improvement of Crystals of Large RNAs by Cation Replacement and Dehydration
journal, September 2014
- Zhang, Jinwei; Ferré-D’Amaré, Adrian R.
- Structure, Vol. 22, Issue 9
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
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
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
Rapid purification of high-activity Taq DNA polymerase
journal, January 1993
- Pluthero, Fred G.
- Nucleic Acids Research, Vol. 21, Issue 20
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
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
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
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
Optimizing splinted ligation of highly structured small RNAs
journal, December 2005
- Kurschat, W. C.
- RNA, Vol. 11, Issue 12
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
In Vitro Transcription: Preparative RNA Yields in Analytical Scale Reactions
journal, August 1994
- Pokrovskaya, I. D.; Gurevich, V. V.
- Analytical Biochemistry, Vol. 220, Issue 2
A simple and efficient method to reduce nontemplated nucleotide addition at the 3′ terminus of RNAs transcribed by T7 RNA polymerase
journal, September 1999
- Kao, C.; Zheng, M.; RÜDisser, S.
- RNA, Vol. 5, Issue 9
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
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
Misincorporation by Wild-Type and Mutant T7 RNA Polymerases: Identification of Interactions That Reduce Misincorporation Rates by Stabilizing the Catalytically Incompetent Open Conformation †
journal, September 2000
- Huang, Jianbin; Brieba, Luis G.; Sousa, Rui
- Biochemistry, Vol. 39, Issue 38
[20] Processing of X-ray diffraction data collected in oscillation mode
book, January 1997
- Otwinowski, Zbyszek; Minor, Wladek
- Macromolecular Crystallography Part A, 307-326
Touchdown PCR for increased specificity and sensitivity in PCR amplification
journal, August 2008
- Korbie, Darren J.; Mattick, John S.
- Nature Protocols, Vol. 3, Issue 9
The preparation of site-specifically modified riboswitch domains as an example for enzymatic ligation of chemically synthesized RNA fragments
journal, August 2008
- Lang, Kathrin; Micura, Ronald
- Nature Protocols, Vol. 3, Issue 9
RNA labeling, conjugation and ligation
journal, June 2011
- Paredes, Eduardo; Evans, Molly; Das, Subha R.
- Methods, Vol. 54, Issue 2
Specific labeling: An effective tool to explore the RNA world
journal, December 2015
- Liu, Yu; Sousa, Rui; Wang, Yun-Xing
- BioEssays, Vol. 38, Issue 2
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
Rapid global structure determination of large RNA and RNA complexes using NMR and small-angle X-ray scattering
journal, October 2010
- Wang, Yun-Xing; Zuo, Xiaobing; Wang, Jinbu
- Methods, Vol. 52, Issue 2
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Efficient labeling of organic molecules using 13 C elemental carbon: universal access to 13 C 2 -labeled synthetic building blocks, polymers and pharmaceuticals
journal, January 2020
- Ledovskaya, Maria S.; Voronin, Vladimir V.; Rodygin, Konstantin S.
- Organic Chemistry Frontiers, Vol. 7, Issue 4
Strategic labelling approaches for RNA single-molecule spectroscopy
journal, April 2019
- Hanspach, Gerd; Trucks, Sven; Hengesbach, Martin
- RNA Biology, Vol. 16, Issue 9
Heavy-atom labeling of RNA by PLOR for de novo crystallographic phasing
journal, April 2019
- Stagno, Jason R.; Yu, Ping; Dyba, Marzena A.
- PLOS ONE, Vol. 14, Issue 4