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

Nature volume 522, pages 368–372 (2015)Cite this article

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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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Figure 1: The PLOR in vitro transcription method.
Figure 2: PLOR isotopical labelling of a 71‐nt RNA.
Figure 3: Application of PLOR in dissecting the co‐existence of multiple conformations using a singly labelled RNA sample.
Figure 4: Application of PLOR‐generated constructs for smFRET.

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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

Authors and Affiliations

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

    Yu Liu, Jinbu Wang, Jason R. Stagno & Yun-Xing Wang

  2. National Institute of Standards and Technology and Department of Chemistry and Biochemistry, JILA, University of Colorado, Boulder, 80309, Colorado, USA

    Erik Holmstrom & David J. Nesbitt

  3. Biochemistry and Biophysics Center, National Heart, Lung and Blood Institute, Bethesda, 20892, Maryland, 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, 21702, Maryland, USA

    Ping Yu & Marzena A. Dyba

  5. Optical Microscopy and Analysis Laboratory, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, 21702, Maryland, USA

    De Chen & Stephen Lockett

  6. Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, 20892, Maryland, USA

    Jinfa Ying

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

    Rui Sousa

Authors
  1. Yu Liu
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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.

Corresponding author

Correspondence to Yun-Xing Wang.

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Extended data figures and tables

Extended Data Figure 1 Synthesis algorithm for the selective labelling of RNA using the PLOR method.

All samples except for fully labelled 71-nt‐CN, S1+Lk1‐CN, S1+Lk1‐H, and 15N‐104-nt‐TCV were synthesized following this algorithm. The initiation, elongation, and termination stages are shown in green, blue, and red, respectively. Various NTP combinations added during the elongation cycles depend on the desired labelling scheme.

Extended Data Figure 2 Automated platform for PLOR synthesis.

a, Diagram of the automated platform for PLOR synthesis depicting its various parts and stations. b, Top‐view (left) and side‐view (right) photographs of the in‐house automated platform.

Extended Data Figure 3 Optimization of experimental conditions for PLOR synthesis.

a, The products of the PLOR (left) and the standard transcription methods (right). PLOR generates a pure full‐length product with the desired labelling in the final step. b, Comparison of PLOR efficiency for Lp2‐CN synthesis under various conditions: I, freshness of DNA‐attached beads; II, anion specificity; III, [Mg2+]; IV, K2SO4 presence in the initiation; V, increasing NTPs (‘1X’ represents NTP amounts in Extended Data Table 1); VI, ratio of ATP/GTP:DNA; VII, ratio of UTP:DNA; and VIII, incubation time of the termination. The right lane contains pure RiboA71 as a control.

Extended Data Figure 4 Comparison of NMR spectra of PLOR‐generated and 71-nt‐CN samples in adenine‐bound form.

Superposition of the 1H13C‐TROSY spectra of 71-nt‐CN with Lp1‐CN (a), Lp2‐CN (b), Lp1+2‐CN (c), Lk2‐CN (d), S1+Lk1‐CN (e), and 4nt‐CN (f). These results indicate that the RNAs synthesized by PLOR have the same fold as 71-nt‐CN generated using the traditional solution‐based transcription method and are functional as evidenced by binding of the adenine ligand.

Extended Data Figure 5 PLOR‐generated riboA71 maintains both sequence and structural fidelity.

a, Structural superposition of the PLOR‐generated riboA71 (PDB accession number 4XNR) with that of the riboA71 generated using the regular in vitro transcription (PDB accession number 4TZX)26. The root mean squared deviation between all C1 atoms was ~ 0.3 Å. b, Structural superposition of the PLOR‐generated riboA71 (PDB accession number 4XNR) with that of the riboA71 (PDB accession number 1Y26)1. The root mean squared deviation between all C1 atoms was ~ 0.4 Å. c, Sequences and secondary structures of the RNAs in b. The arrows denote nucleotide sequence differences between the two ribA71 sequences. d, Composite simulated anneal‐omit 2|Fo| − |Fc| electron density of the riboA71 RNA structure (PDB accession number 4XNR) at 2.2 Å resolution calculated using the final model (1.0 s.d.). e, Portion of the electron density in c in the adenine‐binding pocket unambiguously identifies the nucleobase identities of the PLOR‐generated RNA, revealing no undesired sequence changes introduced by PLOR. f, Portion of the electron density in c of the G6·C66 base pair, which differs in identity between the two structures, suggesting that if there had been undesired sequence substitutions that resulted from using the PLOR method, they would have been readily detected in the crystallographic analysis.

Extended Data Figure 6 Using PLOR to isotopically label a 104‐nt RNA.

a, Secondary structure of the 104‐nt structural element in the TCV genomic RNA. The 15N‐H1‐TCV was synthesized by the PLOR method, and the sequence in red is 15N‐labelled in 15N‐H1‐TCV. b, 15N‐TROSY spectrum of 15N‐H1‐TCV RNA. c, Superposition of the 1H15N‐TROSY spectra of 15N‐H1‐TCV with 15N‐104-nt‐TCV34, indicating that the PLOR‐generated selectively labelled RNA has the same fold as that generated using the regular in vitro transcription.

Extended Data Table 1 PLOR recipes for the RiboA71 and TCV samples
Full size table
Extended Data Table 2 Synthesis efficiencies for the PLOR‐generated NMR samples
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Extended Data Table 3 PLOR recipes for the fluorescent‐labelled RiboA71samples
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Extended Data Table 4 Crystallographic statistics of data collection and refinement for the PLOR‐generated riboA71 structure (PDB accession number 4XNR)
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Liu, Y., Holmstrom, E., Zhang, J. et al. Synthesis and applications of RNAs with position-selective labelling and mosaic composition. Nature 522, 368–372 (2015). https://doi.org/10.1038/nature14352

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