Abstract
Fluorogenic hybridization methods, such as the use of FIT probes, enable the in vivo detection of specific mRNAs transcribed from their endogenous, genetically nonmodified loci. Here, we describe the design, synthesis and injection of nuclease resistant FIT probes into developing Drosophila oocytes to detect endogenous localizing mRNAs as wells as to probe function of structural RNA elements.
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Tyagi S, Kramer FR (1996) Molecular beacons: probes that fluoresce upon hybridization. Nat Biotechnol 14(3):303–308. doi:10.1038/nbt0396-303
Sato S, Watanabe M, Katsuda Y, Murata A, Wang DO, Uesugi M (2015) Live-cell imaging of endogenous mRNAs with a small molecule. Angew Chem 54(6):1855–1858. doi:10.1002/anie.201410339
Oomoto I, Suzuki-Hirano A, Umeshima H, Han YW, Yanagisawa H, Carlton P, Harada Y, Kengaku M, Okamoto A, Shimogori T, Wang DO (2015) ECHO-liveFISH: in vivo RNA labeling reveals dynamic regulation of nuclear RNA foci in living tissues. Nucleic Acids Res 43(19):e126. doi:10.1093/nar/gkv614
Okamoto A (2011) ECHO probes: a concept of fluorescence control for practical nucleic acid sensing. Chem Soc Rev 40(12):5815–5828. doi:10.1039/c1cs15025a
Kohler O, Jarikote DV, Seitz O (2005) Forced intercalation probes (FIT probes): thiazole orange as a fluorescent base in peptide nucleic acids for homogeneous single-nucleotide-polymorphism detection. Chembiochem 6(1):69–77. doi:10.1002/cbic.200400260
Hovelmann F, Gaspar I, Loibl S, Ermilov EA, Roder B, Wengel J, Ephrussi A, Seitz O (2014) Brightness through local constraint--LNA-enhanced FIT hybridization probes for in vivo ribonucleotide particle tracking. Angew Chem 53(42):11370–11375. doi:10.1002/anie.201406022
Ghosh S, Marchand V, Gaspar I, Ephrussi A (2012) Control of RNP motility and localization by a splicing-dependent structure in oskar mRNA. Nat Struct Mol Biol 19(4):441–449. doi:10.1038/nsmb.2257
Zimyanin VL, Belaya K, Pecreaux J, Gilchrist MJ, Clark A, Davis I, St Johnston D (2008) In vivo imaging of oskar mRNA transport reveals the mechanism of posterior localization. Cell 134(5):843–853. doi:10.1016/j.cell.2008.06.053
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Chamiolo, J., Gaspar, I., Ephrussi, A., Seitz, O. (2018). In Vivo Visualization and Function Probing of Transport mRNPs Using Injected FIT Probes. 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_18
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DOI: https://doi.org/10.1007/978-1-4939-7213-5_18
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7212-8
Online ISBN: 978-1-4939-7213-5
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