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

Abstract

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 13C15N-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.

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Figure 1: Diagram of the PLOR synthesis.
Figure 2: PLOR for different RNAs.
Figure 3: Application of PLOR in generating an isotopic-labeled sample for NMR study.
Figure 4: Application of PLOR in generating a fluorescently labeled sample for FRET study.
Figure 5: Application of PLOR in generating a sample labeled with heavy atoms for crystallographic study.

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Acknowledgements

This work was supported by the Intramural Research Programs of the National Cancer Institute (Y.-X.W.). E.H. and D.J.N gratefully acknowledge funding for this work by the National Science Foundation (CHE 1266416 and PHY 1125844) and the National Institute for Standards and Technology. We thank S. Frey from Abteilung Zelluläre Logistik, Max-Planck-Institut für Biophysikalische Chemie, Göttingen, Germany, for providing us with the plasmid for His-tagged Taq DNA polymerase and the preparation protocol, and D. Draper from Johns Hopkins University for providing us with the plasmid for His-tagged T7 RNA polymerase.

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Y.L. performed RNA synthesis, NMR experiments, and X-ray crystallography experiments, and wrote the manuscript; E.H. and D.N. designed and performed smFRET experiments; P.Y. performed enzyme preparation; R.S. provided critical advice on PLOR; J.R.S., K.T. and X.Z. performed X-ray crystallography experiments and data analysis; and Y.-X.W. designed PLOR. All authors revised the manuscript.

Corresponding authors

Correspondence to Yu Liu or Yun-Xing Wang.

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Liu, Y., Holmstrom, E., Yu, P. et al. Incorporation of isotopic, fluorescent, and heavy-atom-modified nucleotides into RNAs by position-selective labeling of RNA. Nat Protoc 13, 987–1005 (2018). https://doi.org/10.1038/nprot.2018.002

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