Abstract
RNA aptamers can serve as valuable tools for studying and manipulating live cells. Fluorescent aptamers are the ones that bind to and turn on fluorescence of small-molecule dyes (fluorogens). Similarly to fluorescent proteins, fluorescent RNA aptamers can be used to image spatial and temporal RNA dynamics in live cells. Additionally, these aptamers can serve as a basis for engineering genetically encoded fluorescent biosensors. This chapter presents a protocol for rapid and efficient screening of RNA aptamer libraries to isolate fluorescent aptamers. The protocol describes how to design, clone, and express RNA aptamer library in bacterial cells and how to screen the bacteria to find aptamers with the desired fluorescent properties.
The original version of this chapter was revised. The erratum to this chapter is available at: DOI 10.1007/978-1-4939-6857-2_27
* Current affiliation: Essen Bioscience, Ann Arbor, MI, USA
Previous affiliation: Department of Pharmacology, Weill Medical College, Cornell University, New York, NY, USA
An erratum to this chapter can be found at http://dx.doi.org/10.1007/978-1-4939-6857-2_27
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References
Famulok M, Hartig JS, Mayer G (2007) Functional aptamers and aptazymes in biotechnology, diagnostics, and therapy. Chem Rev 107:3715–3743
Thiel KW, Giangrande PH (2009) Therapeutic applications of DNA and RNA aptamers. Oligonucleotides 19:209–222
Bruno JG (2015) Predicting the uncertain future of aptamer-based diagnostics and therapeutics. Molecules 20:6866–6887
Kaur G, Roy I (2008) Therapeutic applications of aptamers. Expert Opin Investig Drugs 17:43–60
Keefe AD, Pai S, Ellington A (2010) Aptamers as therapeutics. Nat Rev Drug Discov 9:537–550
Babendure JR, Adams SR, Tsien RY (2003) Aptamers switch on fluorescence of triphenylmethane dyes. J Am Chem Soc 125:14716–14717
Paige JS, Wu KY, Jaffrey SR (2011) RNA mimics of green fluorescent protein. Science 333:642–646
Strack RL, Disney MD, Jaffrey SR (2013) A superfolding Spinach2 reveals the dynamic nature of trinucleotide repeat RNA. Nat Methods 10:1219–1224
Filonov GS, Moon JD, Svensen N, Jaffrey SR (2014) Broccoli: rapid selection of an RNA mimic of green fluorescent protein by fluorescence-based selection and directed evolution. J Am Chem Soc 136(46):16299–16308
Guet D, Burns LT, Maji S, Boulanger J, Hersen P, Wente SR et al (2015) Combining Spinach-tagged RNA and gene localization to image gene expression in live yeast. Nat Commun 6:8882
Zhang J, Fei J, Leslie BJ, Han KY, Kuhlman TE, Ha T (2015) Tandem spinach array for mRNA imaging in living bacterial cells. Sci Rep 5:17295
Lu Z, Filonov GS, Noto JJ, Schmidt CA, Hatkevich TL, Wen Y et al (2015) Metazoan tRNA introns generate stable circular RNAs in vivo. RNA 21:1554–1565
Pothoulakis G, Ceroni F, Reeve B, Ellis T (2014) The spinach RNA aptamer as a characterization tool for synthetic biology. ACS Synth Biol 3:182–187
Paige JS, Nguyen-Duc T, Song W, Jaffrey SR (2012) Fluorescence imaging of cellular metabolites with RNA. Science 335:1194
Song W, Strack RL, Jaffrey SR (2013) Imaging bacterial protein expression using genetically encoded RNA sensors. Nat Methods 10:873–875
You M, Litke JL, Jaffrey SR (2015) Imaging metabolite dynamics in living cells using a spinach-based riboswitch. Proc Natl Acad Sci U S A 112:E2756–E2765
Kellenberger CA, Chen C, Whiteley AT, Portnoy DA, Hammond MC (2015) RNA-based fluorescent biosensors for live cell imaging of second messenger cyclic di-AMP. J Am Chem Soc 137:6432–6435
Kellenberger CA, Wilson SC, Sales-Lee J, Hammond MC (2013) 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
Stoltenburg R, Reinemann C, Strehlitz B (2007) SELEX--a (r)evolutionary method to generate high-affinity nucleic acid ligands. Biomol Eng 24:381–403
Piatkevich KD, Verkhusha VV (2010) Advances in engineering of fluorescent proteins and photoactivatable proteins with red emission. Curr Opin Chem Biol 14:23–29
Stoltenburg R, Nikolaus N, Strehlitz B (2012) Capture-SELEX: selection of DNA aptamers for aminoglycoside antibiotics. J Anal Methods Chem 2012:415697
Koizumi M, Soukup GA, Kerr JN, Breaker RR (1999) Allosteric selection of ribozymes that respond to the second messengers cGMP and cAMP. Nat Struct Biol 6:1062–1071
Strack RL, Song W, Jaffrey SR (2014) Using spinach-based sensors for fluorescence imaging of intracellular metabolites and proteins in living bacteria. Nat Protoc 9:146–155
Gonzales, M.F., Brooks, T., Pukatzki, S.U., and Provenzano, D. (2013) Rapid protocol for preparation of electrocompetent Escherichia coli and Vibrio cholerae. J Vis Exp 80:e50684.
Shao K, Ding W, Wang F, Li H, Ma D, Wang H (2011) Emulsion PCR: a high efficient way of PCR amplification of random DNA libraries in aptamer selection. PLoS One 6:e24910
Song W, Strack RL, Svensen N, Jaffrey SR (2014) Plug-and-play fluorophores extend the spectral properties of Spinach. J Am Chem Soc 136:1198–1201
Lipinski CA, Lombardo F, Dominy BW, Feeney PJ (2001) Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv Drug Deliv Rev 46:3–26
Filonov GS, Kam CW, Song W, Jaffrey SR (2015) In-gel imaging of RNA processing using broccoli reveals optimal aptamer expression strategies. Chem Biol 22:649–660
Acknowledgment
The author thanks Prof. Samie R. Jaffrey for his generous support and scientific guidance. Flow cytometry experiments were performed with the help of J. McCormick and S.Z. Merlin (Department of Pathology and Laboratory Medicine cell sorter core). The author is also grateful to the members of the Jaffrey lab for their feedback as they used this protocol. This work was supported by NIH grants to Prof. Samie R. Jaffrey (R01 NS064516 and R01 EB010249).
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Filonov, G.S. (2017). Rapid Selection of RNA Aptamers that Activate Fluorescence of Small Molecules. In: Tiller, T. (eds) Synthetic Antibodies. Methods in Molecular Biology, vol 1575. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6857-2_17
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DOI: https://doi.org/10.1007/978-1-4939-6857-2_17
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