Abstract
To elucidate the roles, dynamics, and regulation of RNAs, it is vital to be able to visualize the RNA of interest (ROI) in living cells noninvasively. Here, we describe a novel live-cell RNA imaging method using fluorophore- and quencher-binding aptamers, which can be genetically fused to the ROI. In this method, new membrane permeable and nonfluorescent fluorophore–quencher conjugates were utilized, and we showed that their fluorescence increases dramatically upon binding to fluorophore- or quencher-binding aptamers. This phenomenon allowed for labeling the ROI with many different colored fluorophores and also dual-color imaging of two distinct RNAs in live bacteria. Our approach uses small RNA tags and small molecule fluorophores for labeling, thereby minimal perturbation on the function and dynamics of the RNA of interest is expected.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Martin KC, Ephrussi A (2009) mRNA localization: gene expression in the spatial dimension. Cell 136(4):719–730. doi:10.1016/j.cell.2009.01.044
Broude NE (2011) Analysis of RNA localization and metabolism in single live bacterial cells: achievements and challenges. Mol Microbiol 80(5):1137–1147. doi:10.1111/j.1365-2958.2011.07652.x
Montero Llopis P, Jackson AF, Sliusarenko O, Surovtsev I, Heinritz J, Emonet T, Jacobs-Wagner C (2010) Spatial organization of the flow of genetic information in bacteria. Nature 466(7302):77–81. doi:10.1038/nature09152
Keiler KC (2011) RNA localization in bacteria. Curr Opin Microbiol 14(2):155–159. doi:10.1016/j.mib.2011.01.009
Kannaiah S, Amster-Choder O (2016) Methods for studying RNA localization in bacteria. Methods 98:99–103. doi:10.1016/j.ymeth.2015.12.010
Babendure JR, Adams SR, Tsien RY (2003) Aptamers switch on fluorescence of triphenylmethane dyes. J Am Chem Soc 125(48):14716–14717
Constantin TP, Silva GL, Robertson KL, Hamilton TP, Fague K, Waggoner AS, Armitage BA (2008) Synthesis of new fluorogenic cyanine dyes and incorporation into RNA fluoromodules. Org Lett 10(8):1561–1564. doi:10.1021/ol702920e
Lee J, Lee KH, Jeon J, Dragulescu-Andrasi A, Xiao F, Rao J (2010) Combining SELEX screening and rational design to develop light-up fluorophore-RNA aptamer pairs for RNA tagging. ACS Chem Biol 5(11):1065–1074
Holeman LA, Robinson SL, Szostak JW, Wilson C (1998) Isolation and characterization of fluorophore-binding RNA aptamers. Fold Des 3(6):423–431
Carothers JM, Goler JA, Kapoor Y, Lara L, Keasling JD (2010) Selecting RNA aptamers for synthetic biology: investigating magnesium dependence and predicting binding affinity. Nucleic Acids Res 38(8):2736–2747. doi:10.1093/nar/gkq082. gkq082 [pii]
Ellington AD, Szostak JW (1990) In vitro selection of RNA molecules that bind specific ligands. Nature 346(6287):818–822. doi:10.1038/346818a0
Paige JS, KY W, Jaffrey SR (2011) RNA mimics of green fluorescent protein. Science 333(6042):642–646. doi:10.1126/science.1207339
Strack RL, Disney MD, Jaffrey SR (2013) A superfolding Spinach2 reveals the dynamic nature of trinucleotide repeat-containing RNA. Nat Methods 10(12):1219–1224. doi:10.1038/nmeth.2701
Song W, Strack RL, Svensen N, Jaffrey SR (2014) Plug-and-play fluorophores extend the spectral properties of Spinach. J Am Chem Soc 136(4):1198–1201. doi:10.1021/ja410819x
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. doi:10.1021/ja508478x
Dolgosheina EV, Jeng SCY, Panchapakesan SSS, Cojocaru R, Chen PSK, Wilson PD, Hawkins N, Wiggins PA, Unrau PJ (2014) RNA mango aptamer-fluorophore: a bright, high-affinity complex for RNA labeling and tracking. ACS Chem Biol 9(10):2412–2420. doi:10.1021/cb500499x
Sunbul M, Jäschke A (2013) Contact-mediated quenching for RNA imaging in bacteria with a fluorophore-binding aptamer. Angew Chem Int Ed 52(50):13401–13404. doi:10.1002/anie.201306622
Sadhu KK, Mizukami S, Watanabe S, Kikuchi K (2010) Turn-on fluorescence switch involving aggregation and elimination processes for beta-lactamase-tag. Chem Commun (Camb) 46(39):7403–7405. doi:10.1039/c0cc02432e
Sparano BA, Koide K (2005) A strategy for the development of small-molecule-based sensors that strongly fluoresce when bound to a specific RNA. J Am Chem Soc 127(43):14954–14955
Sparano BA, Koide K (2007) Fluorescent sensors for specific RNA: a general paradigm using chemistry and combinatorial biology. J Am Chem Soc 129(15):4785–4794
Murata A, Sato S, Kawazoe Y, Uesugi M (2011) Small-molecule fluorescent probes for specific RNA targets. Chem Commun (Camb) 47(16):4712–4714. doi:10.1039/c1cc10393h
Arora A, Sunbul M, Jäschke A (2015) Dual-colour imaging of RNAs using quencher- and fluorophore-binding aptamers. Nucleic Acids Res 43(21):e144. doi:10.1093/nar/gkv718
Sunbul M, Nacheva L, Jäschke A (2015) Proximity-induced covalent labeling of proteins with a reactive fluorophore-binding peptide tag. Bioconjug Chem 26(8):1466–1469. doi:10.1021/acs.bioconjchem.5b00304
Acknowledgments
This work is supported by Helmholtz Initiative on Synthetic Biology. Murat Sunbul thanks the Alexander von Humboldt Foundation for a postdoctoral fellowship. Ankita Arora thanks the DAAD for a doctoral fellowship.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Sunbul, M., Arora, A., Jäschke, A. (2018). Visualizing RNA in Live Bacterial Cells Using Fluorophore- and Quencher-Binding Aptamers. In: Gaspar, I. (eds) RNA Detection. Methods in Molecular Biology, vol 1649. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7213-5_19
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7213-5_19
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7212-8
Online ISBN: 978-1-4939-7213-5
eBook Packages: Springer Protocols