Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Recurrent RNA motifs as scaffolds for genetically encodable small-molecule biosensors

Nature Chemical Biology volume 13, pages 295–301 (2017)Cite this article

Subjects

Abstract

Allosteric RNA devices are increasingly being viewed as important tools capable of monitoring enzyme evolution, optimizing engineered metabolic pathways, facilitating gene discovery and regulators of nucleic acid–based therapeutics. A key bottleneck in the development of these platforms is the availability of small-molecule-binding RNA aptamers that robustly function in the cellular environment. Although aptamers can be raised against nearly any desired target through in vitro selection, many cannot easily be integrated into devices or do not reliably function in a cellular context. Here, we describe a new approach using secondary- and tertiary-structural scaffolds derived from biologically active riboswitches and small ribozymes. When applied to the neurotransmitter precursors 5-hydroxytryptophan and 3,4-dihydroxyphenylalanine, this approach yielded easily identifiable and characterizable aptamers predisposed for coupling to readout domains to allow engineering of nucleic acid–sensory devices that function in vitro and in the cellular context.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: RNA scaffolds used for selection.
Figure 2: Selection of scaffolded aptamers that selectively bind 5-hydroxytryptophan (5HTP).
Figure 3: Analysis of potential 5HTP aptamers.
Figure 4: In vitro and intracellular performance of 5HTP and L-DOPA sensors.
Figure 5: A 5HTP-aptamer-based biosensor functions in E. coli.

Similar content being viewed by others

Accession codes

Accessions

Protein Data Bank

References

  1. Darmostuk, M., Rimpelova, S., Gbelcova, H. & Ruml, T. Current approaches in SELEX: an update to aptamer selection technology. Biotechnol. Adv. 33, 1141–1161 (2015).

    Article  CAS  PubMed  Google Scholar 

  2. McKeague, M. & Derosa, M.C. Challenges and opportunities for small molecule aptamer development. J. Nucleic Acids 2012, 748913 (2012).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  3. Berens, C., Thain, A. & Schroeder, R. A tetracycline-binding RNA aptamer. Bioorg. Med. Chem. 9, 2549–2556 (2001).

    Article  CAS  PubMed  Google Scholar 

  4. Jenison, R.D., Gill, S.C., Pardi, A. & Polisky, B. High-resolution molecular discrimination by RNA. Science 263, 1425–1429 (1994).

    Article  CAS  PubMed  Google Scholar 

  5. Paige, J.S., Wu, K.Y. & Jaffrey, S.R. RNA mimics of green fluorescent protein. Science 333, 642–646 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Berens, C., Groher, F. & Suess, B. RNA aptamers as genetic control devices: the potential of riboswitches as synthetic elements for regulating gene expression. Biotechnol. J. 10, 246–257 (2015).

    Article  CAS  PubMed  Google Scholar 

  7. Sharma, V., Nomura, Y. & Yokobayashi, Y. Engineering complex riboswitch regulation by dual genetic selection. J. Am. Chem. Soc. 130, 16310–16315 (2008).

    Article  CAS  PubMed  Google Scholar 

  8. Garst, A.D., Edwards, A.L. & Batey, R.T. Riboswitches: structures and mechanisms. Cold Spring Harb. Perspect. Biol. 3, a003533 (2011).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  9. Barrick, J.E. & Breaker, R.R. The distributions, mechanisms, and structures of metabolite-binding riboswitches. Genome Biol. 8, R239 (2007).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  10. Kellenberger, C.A., Wilson, S.C., Sales-Lee, J. & Hammond, M.C. RNA-based fluorescent biosensors for live cell imaging of second messengers cyclic di-GMP and cyclic AMP-GMP. J. Am. Chem. Soc. 135, 4906–4909 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Ketterer, S., Gladis, L., Kozica, A. & Meier, M. Engineering and characterization of fluorogenic glycine riboswitches. Nucleic Acids Res. 44, 5983–5992 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Su, Y., Hickey, S.F., Keyser, S.G. & Hammond, M.C. In vitro and in vivo enzyme activity screening via RNA-based fluorescent biosensors for S-adenosyl-l-homocysteine (SAH). J. Am. Chem. Soc. 138, 7040–7047 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Romanini, D.W., Peralta-Yahya, P., Mondol, V. & Cornish, V.W. A heritable recombination system for synthetic Darwinian evolution in yeast. ACS Synth. Biol. 1, 602–609 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Davis, J.H. & Szostak, J.W. Isolation of high-affinity GTP aptamers from partially structured RNA libraries. Proc. Natl. Acad. Sci. USA 99, 11616–11621 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Filonov, G.S., Moon, J.D., Svensen, N. & Jaffrey, S.R. Broccoli: rapid selection of an RNA mimic of green fluorescent protein by fluorescence-based selection and directed evolution. J. Am. Chem. Soc. 136, 16299–16308 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Peselis, A. & Serganov, A. Structure and function of pseudoknots involved in gene expression control. Wiley Interdiscip. Rev. RNA 5, 803–822 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. de la Peña, M., Dufour, D. & Gallego, J. Three-way RNA junctions with remote tertiary contacts: a recurrent and highly versatile fold. RNA 15, 1949–1964 (2009).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  18. Nakayama, S., Luo, Y., Zhou, J., Dayie, T.K. & Sintim, H.O. Nanomolar fluorescent detection of c-di-GMP using a modular aptamer strategy. Chem. Commun. (Camb.) 48, 9059–9061 (2012).

    Article  CAS  Google Scholar 

  19. Wittmann, A. & Suess, B. Selection of tetracycline inducible self-cleaving ribozymes as synthetic devices for gene regulation in yeast. Mol. Biosyst. 7, 2419–2427 (2011).

    Article  CAS  PubMed  Google Scholar 

  20. Mandal, M., Boese, B., Barrick, J.E., Winkler, W.C. & Breaker, R.R. Riboswitches control fundamental biochemical pathways in Bacillus subtilis and other bacteria. Cell 113, 577–586 (2003).

    Article  CAS  PubMed  Google Scholar 

  21. Sudarsan, N. et al. Riboswitches in eubacteria sense the second messenger cyclic di-GMP. Science 321, 411–413 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Khvorova, A., Lescoute, A., Westhof, E. & Jayasena, S.D. Sequence elements outside the hammerhead ribozyme catalytic core enable intracellular activity. Nat. Struct. Biol. 10, 708–712 (2003).

    Article  CAS  PubMed  Google Scholar 

  23. Majerfeld, I., Puthenvedu, D. & Yarus, M. RNA affinity for molecular l-histidine; genetic code origins. J. Mol. Evol. 61, 226–235 (2005).

    Article  CAS  PubMed  Google Scholar 

  24. Marshall, K.A. & Ellington, A.D. In vitro selection of RNA aptamers. Methods Enzymol. 318, 193–214 (2000).

    Article  CAS  PubMed  Google Scholar 

  25. Illangasekare, M., Turk, R., Peterson, G.C., Lladser, M. & Yarus, M. Chiral histidine selection by d-ribose RNA. RNA 16, 2370–2383 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Wilkinson, K.A., Merino, E.J. & Weeks, K.M. Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE): quantitative RNA structure analysis at single nucleotide resolution. Nat. Protoc. 1, 1610–1616 (2006).

    Article  CAS  PubMed  Google Scholar 

  27. Joyce, G.F. Directed evolution of nucleic acid enzymes. Annu. Rev. Biochem. 73, 791–836 (2004).

    Article  CAS  PubMed  Google Scholar 

  28. Mohr, S. et al. Thermostable group II intron reverse transcriptase fusion proteins and their use in cDNA synthesis and next-generation RNA sequencing. RNA 19, 958–970 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Carothers, J.M., Goler, J.A., Kapoor, Y., Lara, L. & Keasling, J.D. Selecting RNA aptamers for synthetic biology: investigating magnesium dependence and predicting binding affinity. Nucleic Acids Res. 38, 2736–2747 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Ditzler, M.A. et al. High-throughput sequence analysis reveals structural diversity and improved potency among RNA inhibitors of HIV reverse transcriptase. Nucleic Acids Res. 41, 1873–1884 (2013).

    Article  CAS  PubMed  Google Scholar 

  31. Schütze, T. et al. Probing the SELEX process with next-generation sequencing. PLoS One 6, e29604 (2011).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  32. Majerfeld, I. & Yarus, M. A diminutive and specific RNA binding site for l-tryptophan. Nucleic Acids Res. 33, 5482–5493 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Yao, Z., Weinberg, Z. & Ruzzo, W.L. CMfinder: a covariance model based RNA motif finding algorithm. Bioinformatics 22, 445–452 (2006).

    Article  CAS  PubMed  Google Scholar 

  34. Weinberg, Z. & Breaker, R.R. R2R: software to speed the depiction of aesthetic consensus RNA secondary structures. BMC Bioinformatics 12, 3 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Merino, E.J., Wilkinson, K.A., Coughlan, J.L. & Weeks, K.M. RNA structure analysis at single nucleotide resolution by selective 2′-hydroxyl acylation and primer extension (SHAPE). J. Am. Chem. Soc. 127, 4223–4231 (2005).

    Article  CAS  PubMed  Google Scholar 

  36. Stoddard, C.D., Gilbert, S.D. & Batey, R.T. Ligand-dependent folding of the three-way junction in the purine riboswitch. RNA 14, 675–684 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Garst, A.D., Porter, E.B. & Batey, R.T. Insights into the regulatory landscape of the lysine riboswitch. J. Mol. Biol. 423, 17–33 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Batey, R.T., Gilbert, S.D. & Montange, R.K. Structure of a natural guanine-responsive riboswitch complexed with the metabolite hypoxanthine. Nature 432, 411–415 (2004).

    Article  CAS  PubMed  Google Scholar 

  39. Krasilnikov, A.S. & Mondragón, A. On the occurrence of the T-loop RNA folding motif in large RNA molecules. RNA 9, 640–643 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Edwards, T.E. & Ferré-D'Amaré, A.R. Crystal structures of the thi-box riboswitch bound to thiamine pyrophosphate analogs reveal adaptive RNA-small molecule recognition. Structure 14, 1459–1468 (2006).

    Article  CAS  PubMed  Google Scholar 

  41. Serganov, A., Polonskaia, A., Phan, A.T., Breaker, R.R. & Patel, D.J. Structural basis for gene regulation by a thiamine pyrophosphate-sensing riboswitch. Nature 441, 1167–1171 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Nagaswamy, U. & Fox, G.E. Frequent occurrence of the T-loop RNA folding motif in ribosomal RNAs. RNA 8, 1112–1119 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Stojanovic, M.N. & Kolpashchikov, D.M. Modular aptameric sensors. J. Am. Chem. Soc. 126, 9266–9270 (2004).

    Article  CAS  PubMed  Google Scholar 

  44. Ponchon, L. & Dardel, F. Recombinant RNA technology: the tRNA scaffold. Nat. Methods 4, 571–576 (2007).

    Article  CAS  PubMed  Google Scholar 

  45. Kellenberger, C.A., Chen, C., Whiteley, A.T., Portnoy, D.A. & Hammond, M.C. RNA-based fluorescent biosensors for live cell imaging of second messenger cyclic di-AMP. J. Am. Chem. Soc. 137, 6432–6435 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Buck, J., Noeske, J., Wöhnert, J. & Schwalbe, H. Dissecting the influence of Mg2+ on 3D architecture and ligand-binding of the guanine-sensing riboswitch aptamer domain. Nucleic Acids Res. 38, 4143–4153 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Hodak, J.H., Downey, C.D., Fiore, J.L., Pardi, A. & Nesbitt, D.J. Docking kinetics and equilibrium of a GAAA tetraloop-receptor motif probed by single-molecule FRET. Proc. Natl. Acad. Sci. USA 102, 10505–10510 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Caporaso, J.G. et al. QIIME allows analysis of high-throughput community sequencing data. Nat. Methods 7, 335–336 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Edgar, R.C. Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26, 2460–2461 (2010).

    Article  CAS  PubMed  Google Scholar 

  50. Edgar, R.C. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Edwards, A.L., Garst, A.D. & Batey, R.T. Determining structures of RNA aptamers and riboswitches by X-ray crystallography. Methods Mol. Biol. 535, 135–163 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Das, R., Laederach, A., Pearlman, S.M., Herschlag, D. & Altman, R.B. SAFA: semi-automated footprinting analysis software for high-throughput quantification of nucleic acid footprinting experiments. RNA 11, 344–354 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Gilbert, S.D. & Batey, R.T. Monitoring RNA-ligand interactions using isothermal titration calorimetry. Methods Mol. Biol. 540, 97–114 (2009).

    Article  CAS  PubMed  Google Scholar 

  54. Pflugrath, J.W. The finer things in X-ray diffraction data collection. Acta Crystallogr. D Biol. Crystallogr. 55, 1718–1725 (1999).

    Article  CAS  PubMed  Google Scholar 

  55. Adams, P.D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    PubMed  Google Scholar 

  57. Chen, V.B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).

    Article  CAS  PubMed  Google Scholar 

  58. Paige, J.S., Nguyen-Duc, T., Song, W. & Jaffrey, S.R. Fluorescence imaging of cellular metabolites with RNA. Science 335, 1194 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors thank B. Suess (Technical University Darmstadt) and A. Heckel (Goethe University Frankfurt) for the kind gift of α-methyl-5-hydroxy-L-tryptophan, R. Knight and J. Shorenstein for useful discussions about bioinformatics analysis of selections, A. Lambowitz (University of Texas at Austin) for the generous gift of the GsI reverse transcriptase, M. Yarus for the amino acid column labeling protocol, D. McKay and J. Trausch for crystallographic support, and A. Young and A. Palmer for assistance with microscopy. J. Kieft, J. Pfingsten and M. Matyjasik provided critical feedback on the manuscript. This work was supported by grants to R.T.B. from the National Science Foundation (NSF 1150834) and the National Institutes of Health (R01 GM073850) and a predoctoral training grant in Signaling and Cellular Regulation (T32 GM008759).

Author information

Author notes

  1. Ely B Porter & Makenna M Morck

    Present address: Present addresses: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA (E.B.P.) and Department of Biochemistry, Stanford University, Palo Alto, California, USA (M.M.M.).,

Authors and Affiliations

  1. Department of Chemistry and Biochemistry, University of Colorado at Boulder, Boulder, Colorado, USA

    Ely B Porter, Jacob T Polaski, Makenna M Morck & Robert T Batey

Authors
  1. Ely B Porter
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jacob T Polaski
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Makenna M Morck
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Robert T Batey
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

E.B.P. conceived the project and either performed or participated in all experiments and in writing of the manuscript. J.T.P. performed the 5HTP and L-DOPA sensor construction, assay and interpretation. M.M.M. participated in the GR/SSIII selection, screened for crystals of the 5GR-II–5HTP complex and collected crystallographic data. R.T.B. oversaw the project, including planning of experiments, interpretation of data and writing of the manuscript.

Corresponding author

Correspondence to Robert T Batey.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Tables 1–7 and Supplementary Figures 1–14 (PDF 11175 kb)

Supplementary Data

Representative sequences from clusters defining eight 5-hydroxytryptophan binding aptamers (5HTP) and four 3,4-dihydroxyphenylalanine binding aptamers (3,4-DHF). (ZIP 331 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Porter, E., Polaski, J., Morck, M. et al. Recurrent RNA motifs as scaffolds for genetically encodable small-molecule biosensors. Nat Chem Biol 13, 295–301 (2017). https://doi.org/10.1038/nchembio.2278

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.2278

This article is cited by

Search

Advanced 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