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Light-up and FRET aptamer reporters; evaluating their applications for imaging transcription in eukaryotic cells

TitleLight-up and FRET aptamer reporters; evaluating their applications for imaging transcription in eukaryotic cells
Publication TypeJournal Article
Year of Publication2016
AuthorsIlgu, M, Ray, J, Bendickson, L, Wang, TJ, Geraskin, IM, Kraus, G, Nilsen-Hamilton, M
JournalMethods
Volume98
Pagination26-33
Date Published04
Type of ArticleArticle
ISBN Number1046-2023
Accession NumberWOS:000373660100005
Keywordsaptamer, bacteria, binding, containing rna, DFHBI, fluorescence, fluorophore, fret, gene-expression, green, magnesium, Malachite green, Molecular Biology, protein, RNA imaging, sodium
Abstract

aptamers that cause an increase in fluorescence of their ligands when bound by the aptamer have shown promise for reporting on RNA production and localization in vivo. Here we have investigated two light-up aptamers (the malachite green aptamer and the Spinach aptamers) for their suitabilities as reporters of RNA expression in vivo using two eukaryotic cell types, yeast and mammalian. Our analysis focused on the aptamer ligands, their contributions to background noise, and the impact of tandem aptamer strings on signal strength and ligand affinity. Whereas the background fluorescence is very low in vitro, this is not always true for cell imaging. Our results suggest the need for caution in using light up aptamers as reporters for imaging RNA. In particular, images should be collected and analyzed by operators blinded to the sample identities. The appropriate control condition of ligand with the cells in the absence of aptamer expression must be included in each experiment. This control condition establishes that the specific interaction of ligand with aptamer, rather than nonspecific interactions with unknown cell elements, is responsible for the observed fluorescent signals. High background signals due to nonspecific interactions of aptamer ligands with cell components can be minimized by using IMAGEtags (Intracellular Multiaptamer GEnetic tags), which signal by FRET and are promising RNA reporters for imaging transcription. (C) 2015 Elsevier Inc. All rights reserved.

DOI10.1016/j.ymeth.2015.12.009
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Imaging Gene Expression

Short TitleMethods
Alternate JournalMethods