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

Journal Article · · Nature Protocols
 [1];  [2];  [3];  [4];  [4];  [5];  [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)
+ Show Author Affiliations
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.
Research Organization:
Argonne National Lab. (ANL), Argonne, IL (United States)
Sponsoring Organization:
National Institute of Standards and Technology (NIST); National Institutes of Health (NIH), National Cancer Institute; National Science Foundation (NSF); USDOE
Grant/Contract Number:
AC02-06CH11357
OSTI ID:
1461437
Journal Information:
Nature Protocols, Journal Name: Nature Protocols Journal Issue: 5 Vol. 13; ISSN 1754-2189
Publisher:
Nature Publishing GroupCopyright Statement
Country of Publication:
United States
Language:
English

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Cited By (3)

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
Strategic labelling approaches for RNA single-molecule spectroscopy journal April 2019
Heavy-atom labeling of RNA by PLOR for de novo crystallographic phasing journal April 2019

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59 BASIC BIOLOGICAL SCIENCES