Abstract
Photoresponsive artificial riboswitch has the potential to offer a de novo method for spatiotemporal control of gene expression in living cells. Because, even today, it is difficult to design a small molecule binding to a specific RNA sequence, generating an artificial riboswitch that possesses highly specific affinity to a ligand of interest basically depends on in vitro selection procedure where a variety of RNA–ligand complexes can be obtained in established methods. Here, we describe the protocol for in vitro aptamer selection against a photoresponsive peptide ligand containing azobenzene moiety that undergoes photoisomerization through light irradiation. Furthermore, we explain a procedure for surface plasmon resonance assay to detect photoswitchable association and dissociation of RNA–ligand complex on gold surface.
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References
Mandal M, Breaker RR (2004) Gene regulation by riboswitches. Nat Rev Mol Cell Biol 5:451–463
Serganov A, Patel DJ (2007) Ribozymes, riboswitches and beyond: regulation of gene expression without proteins. Nat Rev Genet 8:776–790
Winkler WC, Breaker RR (2005) Regulation of bacterial gene expression by riboswitches. Annu Rev Microbiol 59:487–517
Serganov A, Patel DJ (2012) Metabolite recognition principles and molecular mechanisms underlying riboswitch function. Annu Rev Biophys 41:343–370
Soukup GA, Breaker RR (1999) Engineering precision RNA molecular switches. Proc Natl Acad Sci U S A 96:3584–3589
Werstuck G, Green MR (1998) Controlling gene expression in living cells through small molecule-RNA interactions. Science 282:296–298
Bauer G, Suess B (2006) Engineered riboswitches as novel tools in molecular biology. J Biotechnol 124:4–11
Sinha J, Reyes SJ, Gallivan JP (2010) Reprogramming bacteria to seek and destroy an herbicide. Nat Chem Biol 6:464–470
Suess B, Fink B, Berens C, Stentz R, Hillen W (2004) A theophylline responsive riboswitch based on helix slipping controls gene expression in vivo. Nucleic Acids Res 32:1610–1614
Suess B, Hanson S, Berens C, Fink B, Schroeder R, Hillen W (2003) Conditional gene expression by controlling translation with tetracycline-binding aptamers. Nucleic Acids Res 31:1853–1858
Wilson DS, Szostak JW (1999) In vitro selection of functional nucleic acids. Annu Rev Biochem 68:611–647
Hayashi G, Hagihara M, Dohno C, Nakatani K (2007) Photoregulation of a peptide-RNA interaction on a gold surface. J Am Chem Soc 129:8678–8679
Hayashi G, Hagihara M, Nakatani K (2009) RNA aptamers that reversibly bind photoresponsive azobenzene-containing peptides. Chemistry 15:424–432
Acknowledgements
This work was supported by JSPS grant of Research Fellowship for Young Scientists.
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Hayashi, G., Nakatani, K. (2014). Development of Photoswitchable RNA Aptamer–Ligand Complexes. In: Ogawa, A. (eds) Artificial Riboswitches. Methods in Molecular Biology, vol 1111. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-755-6_3
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DOI: https://doi.org/10.1007/978-1-62703-755-6_3
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Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-754-9
Online ISBN: 978-1-62703-755-6
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