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

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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

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.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

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

References

  1. 1

    Serganov, A. et al. Structural basis for discriminative regulation of gene expression by adenine- and guanine-sensing mRNAs. Chem. Biol. 11, 1729–1741 (2004)

    CAS  PubMed  PubMed Central  Google Scholar 

  2. 2

    Geiger, A., Burgstaller, P., von der Eltz, H., Roeder, A. & Famulok, M. RNA aptamers that bind l-arginine with sub-micromolar dissociation constants and high enantioselectivity. Nucleic Acids Res. 24, 1029–1036 (1996)

    CAS  PubMed  PubMed Central  Google Scholar 

  3. 3

    Mairal, T. et al. Aptamers: molecular tools for analytical applications. Anal. Bioanal. Chem. 390, 989–1007 (2008)

    CAS  PubMed  Google Scholar 

  4. 4

    Shangguan, D. et al. Cell-specific aptamer probes for membrane protein elucidation in cancer cells. J. Proteome Res. 7, 2133–2139 (2008)

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5

    Wilson, C. & Keefe, A. D. Building oligonucleotide therapeutics using non-natural chemistries. Curr. Opin. Chem. Biol. 10, 607–614 (2006)

    CAS  PubMed  Google Scholar 

  6. 6

    Usman, N., Ogilvie, K. K., Jiang, M. Y. & Cedergen, R. J. Automated chemical synthesis of long oligoribonucleotides using 2′-O-silylated ribonucleoside 3′-O-phosphoramidites on a controlled-pore glass support: synthesis of a 43-nucleotide sequence similar to the 3′-half molecule of an Escherichia coli formyl-methionine tRNA. J. Am. Chem. Soc. 109, 7845–7854 (1987)

    CAS  Google Scholar 

  7. 7

    Scaringe, S. A., Wincott, F. E. & Caruthers, M. H. Novel RNA synthesis method using 5′-O-silyl-2′-O-orthorster protection groups. J. Am. Chem. Soc. 120, 11820–11821 (1998)

    CAS  Google Scholar 

  8. 8

    Milligan, J. F., Groebe, D. R., Witherell, G. W. & Uhlenbeck, O. C. Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates. Nucleic Acids Res. 15, 8783–8798 (1987)

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9

    Lu, K., Miyazaki, Y. & Summers, M. F. Isotope labeling strategies for NMR studies of RNA. J. Biomol. NMR 46, 113–125 (2010)

    CAS  PubMed  Google Scholar 

  10. 10

    Lyakhov, D. L. et al. Pausing and termination by bacteriophage T7 RNA polymerase. J. Mol. Biol. 280, 201–213 (1998)

    CAS  PubMed  Google Scholar 

  11. 11

    Guo, Q., Nayak, D., Brieba, L. G. & Sousa, R. Major conformational changes during T7RNAP transcription initiation coincide with, and are required for, promoter release. J. Mol. Biol. 353, 256–270 (2005)

    CAS  PubMed  Google Scholar 

  12. 12

    Sohn, Y., Shen, H. & Kang, C. Stepwise walking and cross-linking of RNA with elongating T7 RNA polymerase. Methods Enzymol. 371, 170–179 (2003)

    CAS  PubMed  Google Scholar 

  13. 13

    Nudler, E., Gusarov, I. & Bar-Nahum, G. Methods of walking with the RNA polymerase. Methods Enzymol. 371, 160–169 (2003)

    CAS  PubMed  Google Scholar 

  14. 14

    Pavlov, M. Y., Freistroffer, D. V. & Ehrenberg, M. Synthesis of region-labelled proteins for NMR studies by in vitro translation of column-coupled mRNAs. Biochimie 79, 415–422 (1997)

    CAS  PubMed  Google Scholar 

  15. 15

    Marble, H. A. & Davis, R. H. RNA transcription from immobilized DNA templates. Biotechnol. Prog. 11, 393–396 (1995)

    CAS  PubMed  Google Scholar 

  16. 16

    Huang, J., Brieba, L. G. & Sousa, R. Misincorporation by wild-type and mutant T7 RNA polymerases: identification of interactions that reduce misincorporation rates by stabilizing the catalytically incompetent open conformation. Biochemistry 39, 11571–11580 (2000)

    CAS  PubMed  Google Scholar 

  17. 17

    Makarova, O. V., Makarov, E. M., Sousa, R. & Dreyfus, M. Transcribing of Escherichia coli genes with mutant T7 RNA polymerases: stability of lacZ mRNA inversely correlates with polymerase speed. Proc. Natl Acad. Sci. USA 92, 12250–12254 (1995)

    ADS  CAS  PubMed  Google Scholar 

  18. 18

    Mentesana, P. E., Chin-Bow, S. T., Sousa, R. & McAllister, W. T. Characterization of halted T7 RNA polymerase elongation complexes reveals multiple factors that contribute to stability. J. Mol. Biol. 302, 1049–1062 (2000)

    CAS  PubMed  Google Scholar 

  19. 19

    Leipply, D. & Draper, D. E. Dependence of RNA tertiary structural stability on Mg2+ concentration: interpretation of the Hill equation and coefficient. Biochemistry 49, 1843–1853 (2010)

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 20

    Delfosse, V. et al. Riboswitch structure: an internal residue mimicking the purine ligand. Nucleic Acids Res. 38, 2057–2068 (2010)

    CAS  PubMed  Google Scholar 

  21. 21

    Noeske, J. et al. An intermolecular base triple as the basis of ligand specificity and affinity in the guanine- and adenine-sensing riboswitch RNAs. Proc. Natl Acad. Sci. USA 102, 1372–1377 (2005)

    ADS  CAS  PubMed  Google Scholar 

  22. 22

    Lee, M. K., Gal, M., Frydman, L. & Varani, G. Real-time multidimensional NMR follows RNA folding with second resolution. Proc. Natl Acad. Sci. USA 107, 9192–9197 (2010)

    ADS  CAS  PubMed  Google Scholar 

  23. 23

    Lemay, J. F., Penedo, J. C., Tremblay, R., Lilley, D. M. & Lafontaine, D. A. Folding of the adenine riboswitch. Chem. Biol. 13, 857–868 (2006)

    CAS  PubMed  Google Scholar 

  24. 24

    Dalgarno, P. A. et al. Single-molecule chemical denaturation of riboswitches. Nucleic Acids Res. 41, 4253–4265 (2013)

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 25

    Rieder, R., Lang, K., Graber, D. & Micura, R. Ligand-induced folding of the adenosine deaminase A-riboswitch and implications on riboswitch translational control. ChemBioChem 8, 896–902 (2007)

    CAS  PubMed  Google Scholar 

  26. 26

    Zhang, J. & Ferré-D'Amaré, A. R. Dramatic improvement of crystals of large RNAs by cation replacement and dehydration. Structure 22, 1363–1371 (2014)

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 27

    Zhang, Q., Stelzer, A. C., Fisher, C. K. & Al-Hashimi, H. M. Visualizing spatially correlated dynamics that directs RNA conformational transitions. Nature 450, 1263–1267 (2007)

    ADS  CAS  PubMed  Google Scholar 

  28. 28

    Leipply, D. & Draper, D. E. Effects of Mg2+ on the free energy landscape for folding a purine riboswitch RNA. Biochemistry 50, 2790–2799 (2011)

    CAS  PubMed  PubMed Central  Google Scholar 

  29. 29

    Ferré-D'Amaré, A. R. & Doudna, J. A. Use of cis- and trans-ribozymes to remove 5′ and 3′ heterogeneities from milligrams of in vitro transcribed RNA. Nucleic Acids Res. 24, 977–978 (1996)

    PubMed  PubMed Central  Google Scholar 

  30. 30

    Pinheiro, V. B. & Holliger, P. Towards XNA nanotechnology: new materials from synthetic genetic polymers. Trends Biotechnol. 32, 321–328 (2014)

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31

    Korbie, D. J. & Mattick, J. S. Touchdown PCR for increased specificity and sensitivity in PCR amplification. Nature Protocols 3, 1452–1456 (2008)

    CAS  PubMed  Google Scholar 

  32. 32

    Kao, C., Zheng, M. & Rüdisser, S. A simple and efficient method to reduce nontemplated nucleotide addition at the 3 terminus of RNAs transcribed by T7 RNA polymerase. RNA 5, 1268–1272 (1999)

    CAS  PubMed  PubMed Central  Google Scholar 

  33. 33

    Mukherjee, S., Brieba, L. G. & Sousa, R. Structural transitions mediating transcription initiation by T7 RNA polymerase. Cell 110, 81–91 (2002)

    CAS  PubMed  Google Scholar 

  34. 34

    Zuo, X. et al. Solution structure of the cap-independent translational enhancer and ribosome-binding element in the 3′ UTR of turnip crinkle virus. Proc. Natl Acad. Sci. USA 107, 1385–1390 (2010)

    ADS  CAS  PubMed  Google Scholar 

  35. 35

    Ying, J. et al. Measurement of 1H-15N and 1H-13C residual dipolar couplings in nucleic acids from TROSY intensities. J. Biomol. NMR 51, 89–103 (2011)

    CAS  PubMed  PubMed Central  Google Scholar 

  36. 36

    Delaglio, F. et al. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277–293 (1995)

    CAS  Google Scholar 

  37. 37

    Holmstrom, E. D. & Nesbitt, D. J. Single-molecule fluorescence resonance energy transfer studies of the human telomerase RNA pseudoknot: temperature-/urea-dependent folding kinetics and thermodynamics. J. Phys. Chem. B 118, 3853–3863 (2014)

    CAS  PubMed  PubMed Central  Google Scholar 

  38. 38

    Kapanidis, A. N. et al. Alternating-laser excitation of single molecules. Acc. Chem. Res. 38, 523–533 (2005)

    CAS  PubMed  Google Scholar 

  39. 39

    Aitken, C. E., Marshall, R. A. & Puglisi, J. D. An oxygen scavenging system for improvement of dye stability in single-molecule fluorescence experiments. Biophys. J. 94, 1826–1835 (2008)

    CAS  PubMed  PubMed Central  Google Scholar 

  40. 40

    Cordes, T., Vogelsang, J. & Tinnefeld, P. On the mechanism of Trolox as antiblinking and antibleaching reagent. J. Am. Chem. Soc. 131, 5018–5019 (2009)

    CAS  PubMed  Google Scholar 

  41. 41

    Fiore, J. L., Hodak, J. H., Piestert, O., Downey, C. D. & Nesbitt, D. J. Monovalent and divalent promoted GAAA tetraloop-receptor tertiary interactions from freely diffusing single-molecule studies. Biophys. J. 95, 3892–3905 (2008)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  42. 42

    Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010)

    CAS  PubMed  PubMed Central  Google Scholar 

  43. 43

    Afonine, P. V. et al. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D 68, 352–367 (2012)

    CAS  Google Scholar 

  44. 44

    Steinberg, G., Stromsborg, K., Thomas, L., Barker, D. & Zhao, C. Strategies for covalent attachment of DNA to beads. Biopolymers 73, 597–605 (2004)

    CAS  PubMed  Google Scholar 

  45. 45

    Chivers, C. E. et al. A streptavidin variant with slower biotin dissociation and increased mechanostability. Nature Methods 7, 391–393 (2010)

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

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

Authors

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.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

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
Extended Data Table 2 Synthesis efficiencies for the PLOR‐generated NMR samples
Extended Data Table 3 PLOR recipes for the fluorescent‐labelled RiboA71samples
Extended Data Table 4 Crystallographic statistics of data collection and refinement for the PLOR‐generated riboA71 structure (PDB accession number 4XNR)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing