Letter | Published:

Synthesis and applications of RNAs with position-selective labelling and mosaic composition

Nature volume 522, pages 368372 (18 June 2015) | Download Citation

Abstract

Knowledge of the structure and dynamics of RNA molecules is critical to understanding their many biological functions. Furthermore, synthetic RNAs have applications as therapeutics and molecular sensors. Both research and technological applications of RNA would be dramatically enhanced by methods that enable incorporation of modified or labelled nucleotides into specifically designated positions or regions of RNA. However, the synthesis of tens of milligrams of such RNAs using existing methods has been impossible. Here we develop a hybrid solid–liquid phase transcription method and automated robotic platform for the synthesis of RNAs with position-selective labelling. We demonstrate its use by successfully preparing various isotope- or fluorescently labelled versions of the 71-nucleotide aptamer domain of an adenine riboswitch1 for nuclear magnetic resonance spectroscopy or single-molecule Förster resonance energy transfer, respectively. Those RNAs include molecules that were selectively isotope-labelled in specific loops, linkers, a helix, several discrete positions, or a single internal position, as well as RNA molecules that were fluorescently labelled in and near kissing loops. These selectively labelled RNAs have the same fold as those transcribed using conventional methods, but they greatly simplify the interpretation of NMR spectra. The single-position isotope- and fluorescently labelled RNA samples reveal multiple conformational states of the adenine riboswitch. Lastly, we describe a robotic platform and the operation that automates this technology. Our selective labelling method may be useful for studying RNA structure and dynamics and for making RNA sensors for a variety of applications including cell-biological studies, substance detection2, and disease diagnostics3,4.

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Accessions

Primary accessions

Protein Data Bank

Data deposits

Structure coordinates have been deposited in Protein Data Bank under accession number 4XNR.

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Acknowledgements

We thank D. E. Draper, A. Bax, A. Byrd, M. Summers, A. Rein, and J. Strathern for discussions. This work was supported in part by the Intramural Research Programs of the National Cancer Institute, the National Institute of Diabetes, Digestive and Kidney Diseases, the National Heart, Lung and Blood Institute; by the Intramural Antiviral Target Program (IATAP) of the Office of the Director, National Institutes of Health; by the 2013 Director’s Challenge Innovation Award of the National Institutes of Health; by the fund from the National Cancer Institute under contract number HHSN261200800001E; by the National Science Foundation (CHE1266416, PHYS1125844); by the National Institutes of Health Molecular Biophysics Training Program (T32 GM-065103); and by the W. M. Keck Foundation, and the National Institute of Standards and Technology. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government.

Author information

Affiliations

  1. Protein-Nucleic Acid Interaction Section, Structural Biophysics Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, Maryland 21702, USA

    • Yu Liu
    • , Jinbu Wang
    • , Jason R. Stagno
    •  & Yun-Xing Wang
  2. JILA, National Institute of Standards and Technology and Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309, USA

    • Erik Holmstrom
    •  & David J. Nesbitt
  3. Biochemistry and Biophysics Center, National Heart, Lung and Blood Institute, Bethesda, Maryland 20892, USA

    • Jinwei Zhang
    •  & Adrian R. Ferré-D’Amaré
  4. Structural Biophysics Laboratory, Basic Science Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702, USA

    • Ping Yu
    •  & Marzena A. Dyba
  5. Optical Microscopy and Analysis Laboratory, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702, USA

    • De Chen
    •  & Stephen Lockett
  6. Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA

    • Jinfa Ying
  7. Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78229, USA

    • Rui Sousa

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Contributions

Y.L. and P.Y. performed RNA synthesis and NMR experiments; E.H. and D.J.N. designed and performed smFRET experiments; J.W. contributed to chemical shift assignments; J.Z. and A.R.F. helped to design the DNA template using PCR, crystallized and determined the three-dimensional structure of the PLOR-generated riboA71; J.Y., M.A.D., D.C., and S.L. helped to characterize RNA; R.S. provided critical advice about T7-enzymatic synthesis and revised the manuscript; J.R.S. helped to characterize RNAs and revised the manuscript; Y.L., E.H., J.Z., J.R.S., and Y.-X.W. prepared figures; Y.-X.W. designed PLOR and the automated platform and wrote the manuscript. All authors discussed the results.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Yun-Xing Wang.

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DOI

https://doi.org/10.1038/nature14352

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