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In vitro selection of signaling aptamers

Nature Biotechnology volume 18, pages 1293–1297 (2000)Cite this article

Abstract

Reagentless biosensors that can directly transduce molecular recognition to optical signals should potentiate the development of sensor arrays for a wide variety of analytes. Nucleic acid aptamers that bind ligands tightly and specifically can be readily selected, but may prove difficult to adapt to biosensor applications. We have therefore attempted to develop selection methods that couple the broad molecular recognition properties of aptamers with signal transduction. Anti-adenosine aptamers were selected from a pool that was skewed to contain very few fluoresceinated uridines. The primary family of aptamers showed a doubling of relative fluorescence intensity at saturating concentrations of a cognate analyte, ATP, and could sense ATP concentrations as low as 25 μM. A single uridine was present in the best signaling aptamer. Surprisingly, other dyes could substitute for fluorescein and still specifically signal the presence of ATP, indicating that the single uridine functioned as a general “switch” for transducing molecular recognition to optical signals.

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Figure 1: Selection of signaling aptamers.
Figure 2: Mapping functional uridine residues.
Figure 3: Signaling specificity of rafl7-U61C.
Figure 4: Response curves for signaling aptamers in analyte mixtures.
Figure 5: Signaling with different fluorophores.
Figure 6: Response curves for selected (rafl7s) and designed (RNA-13-AC) signaling aptamers.

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References

  1. Hesselberth, J., Robertson, M.P., Jhaveri, S.D. & Ellington, A.D. In vitro selection of nucleic acids for diagnostic applications. Rev. Mol. Biotechnol. 74, 15–25 (2000).

    Article  CAS  Google Scholar 

  2. Jayasena,S.D. Aptamers: an emerging class of molecules that rival antibodies in diagnostics . Clin. Chem. 45, 1628– 1650 (1999)

    CAS  PubMed  Google Scholar 

  3. Famulok, M. & Jenne, A. Oligonucleotide libraries—variatio delectat. Curr. Opin. Chem. Biol. 2, 320 –327 (1998).

    Article  CAS  PubMed  Google Scholar 

  4. Gold, L., Polisky, B., Uhlenbeck, O. & Yarus, M. Diversity of oligonucleotide functions. Annu. Rev. Biochem. 64, 763–797 (1995).

    Article  CAS  PubMed  Google Scholar 

  5. Osborne, S.E., Matsumura, I. & Ellington, A.D. Aptamers as therapeutic and diagnostic reagents: problems and prospects. Curr. Opin. Chem. Biol. 1, 5–9 (1997).

    Article  CAS  PubMed  Google Scholar 

  6. Famulok, M. & Mayer, G. Aptamers as tools in molecular biology and immunology. Curr. Top. Microbiol. Immunol. 243, 123–136 ( 1999).

    CAS  PubMed  Google Scholar 

  7. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Morris, K.N., Jensen, K.B., Julin, C.M., Weil, M. & Gold, L. High affinity ligands from in vitro selection: complex targets. Proc. Natl. Acad. Sci. USA 95, 2902–2907 (1998).

    Article  CAS  PubMed  Google Scholar 

  9. German, I., Buchanan, D.D. & Kennedy, R.T. Aptamers as ligands in affinity probe capillary electrophoresis . Anal. Chem. 70, 4540– 4545 (1998).

    Article  CAS  PubMed  Google Scholar 

  10. Potyrailo, R.A., Conrad, R.C., Ellington, A.D. & Hieftje, G.M. Adapting selected nucleic acid ligands (aptamers) to biosensors . Anal. Chem. 70, 3419– 3425 (1998).

    Article  CAS  PubMed  Google Scholar 

  11. Davis, K.A., Lin, Y., Abrams, B. & Jayasena, S.D. Staining of cell surface human CD4 with 2′-F-pyrimidine-containing RNA aptamers for flow cytometry. Nucleic Acids Res. 26, 3915–3924 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Davis, K.A., Abrams, B., Lin, Y. & Jayasena, S.D. Use of a high affinity DNA ligand in flow cytometry. Nucleic Acids Res. 24, 702–706 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Giuliano, K.A. & Taylor, D.L. Fluorescent-protein biosensors: new tools for drug discovery. Trends Biotechnol. 16, 135–140 (1998).

    Article  CAS  PubMed  Google Scholar 

  14. Hirshberg, M. et al. Crystal structure of phosphate binding protein labeled with a coumarin fluorophore, a probe for inorganic phosphate. Biochemistry 37, 10381–10385 (1998).

    Article  CAS  PubMed  Google Scholar 

  15. Brune, M. et al. Mechanism of inorganic phosphate interaction with phosphate binding protein from Escherichia coli. Biochemistry 37, 10370–10380 (1998).

    Article  CAS  PubMed  Google Scholar 

  16. Gilardi, G., Zhou, L.Q., Hibbert, L. & Cass, A.E. Engineering the maltose binding protein for reagentless fluorescence sensing. Anal. Chem. 66, 3840–3847 (1994).

    Article  CAS  PubMed  Google Scholar 

  17. Marvin, J.S. et al. The rational design of allosteric interactions in a monomeric protein and its applications to the construction of biosensors. Proc. Natl. Acad. Sci. USA 94, 4366–4371 (1997).

    Article  CAS  PubMed  Google Scholar 

  18. Adams, S.R., Harootunian, A.T., Buechler, Y.J., Taylor, S.S. & Tsien, R.Y. Fluorescence ratio imaging of cyclic AMP in single cells. Nature 349, 694–697 (1991).

    Article  CAS  PubMed  Google Scholar 

  19. Patel, D.J. et al. Structure, recognition and adaptive binding in RNA aptamer complexes . J. Mol. Biol. 272, 645– 664 (1997).

    Article  CAS  PubMed  Google Scholar 

  20. Westhof, E. & Patel, D.J. Nucleic acids. From self-assembly to induced-fit recognition. Curr. Opin. Struct. Biol. 7, 305–309 ( 1997).

    Article  CAS  PubMed  Google Scholar 

  21. Jhaveri, S.D. et al. Designed signaling aptamers that transduce molecular recognition to changes in fluorescence intensity. J. Am. Chem. Soc. 122, 2469–2473 (2000)

    Article  CAS  Google Scholar 

  22. Rogers, J. & Joyce, G.F. A ribozyme that lacks cytidine. Nature 402, 323– 325 (1999).

    Article  CAS  Google Scholar 

  23. Sassanfar, M. & Szostak, J.W. An RNA motif that binds ATP. Nature 364, 550– 553 (1993).

    Article  CAS  PubMed  Google Scholar 

  24. Crick, F.H. The origin of the genetic code. J. Mol. Biol. 38, 367–379 (1968).

    Article  CAS  Google Scholar 

  25. Wachtershauser, G. An all-purine precursor of nucleic acids. Proc. Natl. Acad. Sci. USA 85, 1134–1135 ( 1988).

    Article  CAS  PubMed  Google Scholar 

  26. Burke, D.H. & Gold, L. RNA aptamers to the adenosine moiety of S-adenosyl methionine: structural inferences from variations on a theme and the reproducibility of SELEX. Nucleic Acids Res. 25, 2020–2024 ( 1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Burgstaller, P. & Famulok, M. Isolation of RNA-aptamers for biological cofactors by in vitro selection. Angew. Chem. Int. Edn. Engl. 33, 1084–1087 (1994)

    Article  Google Scholar 

  28. Ellington, A.D., Hesselberth, J., Jhaveri, S. & Robertson, M.P. Combinatorial methods: aptamers and aptazymes Proc. SPIE 3858, 126–134 (1999)

    Article  CAS  Google Scholar 

  29. Taylor, L.C. & Walt, D.R. Application of high density optical microwell arrays in a live-cell biosensing system Anal. Biochem. 278, 132–142 (2000)

    Article  CAS  PubMed  Google Scholar 

  30. Walt, D.R. Techview: molecular biology. Bead-based fiber-optic arrays Science 287, 451–452 ( 2000).

    Article  CAS  PubMed  Google Scholar 

  31. Savoy, S. et al. Solution based analysis of multiple analytes by a sensor array: toward the development of an “electronic tongue.” Proc. SPIE 3539, 17–26 (1998)

    Article  CAS  Google Scholar 

  32. Lavigne, J.J. et al. Single-analyte to multianalyte fluorescence sensors Proc. SPIE 3602, 220–231 ( 1999).

    Article  CAS  Google Scholar 

  33. Pollard, J., Bell, S. & Ellington, A. Design, synthesis, and amplification of DNA pools for in vitro selection, In Current protocols in nucleic acid chemistry (ed. Beaucage, S.) 9.2.1–9.2.23 (Wiley, New York, NY; 2000).

    Google Scholar 

  34. Gallagher, S. Quantitation of DNA and RNA with absorption and fluorescence spectroscopy In Current protocols in molecular biology. (ed. Ausubel, F.) A.3D.1-A.3D.8 (Wiley, New York, NY; 2000 ).

    Google Scholar 

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Acknowledgements

This work was supported by a grant from Foundation for Research.

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry, University of Texas at Austin, Austin, 78712, TX

    Sulay Jhaveri, Manjula Rajendran & Andrew D. Ellington

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Correspondence to Andrew D. Ellington.

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Jhaveri, S., Rajendran, M. & Ellington, A. In vitro selection of signaling aptamers. Nat Biotechnol 18, 1293–1297 (2000). https://doi.org/10.1038/82414

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