Abstract
Artificial riboswitches that respond to the concentrations of intracellular proteins are a promising tool with a variety of applications. They can be designed and engineered using existing RNA aptamers that target proteins. Aptamers are generated via an iterative selection–amplification process, known as systematic evolution of ligands by exponential enrichment (SELEX). This chapter describes a SELEX procedure for the identification of RNA aptamers against hexa-histidine-tagged proteins. For the efficient enrichment of higher affinity aptamers, the selection stringency should be gradually increased. Undesired RNA species that bind to affinity resins can be eliminated from the pool by using a negative selection step and alternating different types of resins.
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References
Ausländer S, Fussenegger M (2013) From gene switches to mammalian designer cells: present and future prospects. Trends Biotechnol 31:155–168
Ellington AD, Chen X, Robertson M, Syrett A (2009) Evolutionary origins and directed evolution of RNA. Int J Biochem Cell Biol 41:254–265
Joyce GF (2007) Forty years of in vitro evolution. Angew Chem Int Ed Engl 46:6420–6436
Ellington A, Szostak JW (1990) In vitro selection of RNA molecules that bind specific ligands. Nature 346:818–822
Tuerk C, Gold L (1990) Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249:505–510
Klussmann S (ed) (2006) The aptamer handbook: functional oligonucleotides and their applications. Wiley-VCH, Weinheim
Stoltenburg R, Reinemann C, Strehlitz B (2007) SELEX—a (r)evolutionary method to generate high-affinity nucleic acid ligands. Biomol Eng 24:381–403
Milligan JF, Groebe DR, Witherell GW, Uhlenbeck OC (1987) Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates. Nucleic Acids Res 15:8783–8798
Di Primo C, Dausse E, Toulmé JJ (2011) Surface Plasmon resonance investigation of RNA aptamer-RNA ligand interactions. Methods Mol Biol 764:279–300
Ishino T, Atarashi K, Uchiyama S, Yamami T, Saihara Y, Yoshida T, Hara H, Yokose K, Kobayashi Y, Nakamura Y (2000) Interaction of ribosome recycling factor and elongation factor EF-G with E. coli ribosomes studied by the surface plasmon resonance technique. Genes Cells 5:953–963
Werner A, Hahn U (2009) Fluorescence correlation spectroscopy (FCS)-based characterisation of aptamer ligand interaction. Methods Mol Biol 535:107–114
Schürer H, Buchynskyy A, Korn K, Famulok M, Welzei P, Hahn U (2001) Fluorescence correlation spectroscopy as a new method for the investigation of aptamer/target interactions. Biol Chem 382:479–481
Ohuchi S, Mori Y, Nakamura Y (2012) Evolution of an inhibitory RNA aptamer against T7 RNA polymerase. FEBS Open Bio 2:203–207
Ekland EH, Bartel DP (1995) The secondary structure and sequence optimization of an RNA ligase ribozyme. Nucleic Acids Res 23:3231–3238
Sabeti PC, Unrau PJ, Bartel DP (1997) Accessing rare activities from random RNA sequences: the importance of the length of molecules in the starting pool. Chem Biol 4:767–774
Hellman LM, Fried MG (2007) Electrophoretic mobility shift assay (EMSA) for detecting protein-nucleic acid interactions. Nat Protoc 2:1849–1861
Li M, Su ZG, Janson JC (2004) In vitro protein refolding by chromatographic procedures. Protein Expr Purif 33:1–10
Zhang YJ, Pan HY, Gao SJ (2001) Reverse transcription slippage over the mRNA secondary structure of the LIP1 gene. Biotechniques 31:1286–1294
Breaker RR, Joyce GF (1993) Minimonsters: evolutionary byproducts of in vitro RNA amplification. In: Fleischaker GR, Colonna S, Luisi PL (eds) Proceedings of the NATO conference on self-reproduction of supramolecular structures. Kluwer Academic Press, Dordrecht, pp 127–135
Seetin MG, Mathews DH (2012) RNA structure prediction: an overview of methods. Methods Mol Biol 905:99–122
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Ohuchi, S. (2014). Identification of RNA Aptamers Against Recombinant Proteins with a Hexa-Histidine Tag. 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_4
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DOI: https://doi.org/10.1007/978-1-62703-755-6_4
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