Concatemeric Broccoli reduces mRNA stability and induces aggregates.

Fluorogenic aptamers are an alternative to established methodology for real-time imaging of RNA transport and dynamics. We developed Broccoli-aptamer concatemers ranging from 4 to 128 substrate-binding site repeats and characterized their behavior fused to an mCherry-coding mRNA in transient transfe...

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Autores principales:	Marco R Rink, Marisa A P Baptista, Felix J Flomm, Thomas Hennig, Adam W Whisnant, Natalia Wolf, Jürgen Seibel, Lars Dölken, Jens B Bosse
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/3179583d0e394143b1db375ef4588a2b
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spelling	oai:doaj.org-article:3179583d0e394143b1db375ef4588a2b2021-12-02T20:15:16ZConcatemeric Broccoli reduces mRNA stability and induces aggregates.1932-620310.1371/journal.pone.0244166https://doaj.org/article/3179583d0e394143b1db375ef4588a2b2021-01-01T00:00:00Zhttps://doi.org/10.1371/journal.pone.0244166https://doaj.org/toc/1932-6203Fluorogenic aptamers are an alternative to established methodology for real-time imaging of RNA transport and dynamics. We developed Broccoli-aptamer concatemers ranging from 4 to 128 substrate-binding site repeats and characterized their behavior fused to an mCherry-coding mRNA in transient transfection, stable expression, and in recombinant cytomegalovirus infection. Concatemerization of substrate-binding sites increased Broccoli fluorescence up to a concatemer length of 16 copies, upon which fluorescence did not increase and mCherry signals declined. This was due to the combined effects of RNA aptamer aggregation and reduced RNA stability. Unfortunately, both cellular and cytomegalovirus genomes were unable to maintain and express high Broccoli concatemer copy numbers, possibly due to recombination events. Interestingly, negative effects of Broccoli concatemers could be partially rescued by introducing linker sequences in between Broccoli repeats warranting further studies. Finally, we show that even though substrate-bound Broccoli is easily photobleached, it can still be utilized in live-cell imaging by adapting a time-lapse imaging protocol.Marco R RinkMarisa A P BaptistaFelix J FlommThomas HennigAdam W WhisnantNatalia WolfJürgen SeibelLars DölkenJens B BossePublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 16, Iss 8, p e0244166 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Marco R Rink Marisa A P Baptista Felix J Flomm Thomas Hennig Adam W Whisnant Natalia Wolf Jürgen Seibel Lars Dölken Jens B Bosse Concatemeric Broccoli reduces mRNA stability and induces aggregates.
description	Fluorogenic aptamers are an alternative to established methodology for real-time imaging of RNA transport and dynamics. We developed Broccoli-aptamer concatemers ranging from 4 to 128 substrate-binding site repeats and characterized their behavior fused to an mCherry-coding mRNA in transient transfection, stable expression, and in recombinant cytomegalovirus infection. Concatemerization of substrate-binding sites increased Broccoli fluorescence up to a concatemer length of 16 copies, upon which fluorescence did not increase and mCherry signals declined. This was due to the combined effects of RNA aptamer aggregation and reduced RNA stability. Unfortunately, both cellular and cytomegalovirus genomes were unable to maintain and express high Broccoli concatemer copy numbers, possibly due to recombination events. Interestingly, negative effects of Broccoli concatemers could be partially rescued by introducing linker sequences in between Broccoli repeats warranting further studies. Finally, we show that even though substrate-bound Broccoli is easily photobleached, it can still be utilized in live-cell imaging by adapting a time-lapse imaging protocol.
format	article
author	Marco R Rink Marisa A P Baptista Felix J Flomm Thomas Hennig Adam W Whisnant Natalia Wolf Jürgen Seibel Lars Dölken Jens B Bosse
author_facet	Marco R Rink Marisa A P Baptista Felix J Flomm Thomas Hennig Adam W Whisnant Natalia Wolf Jürgen Seibel Lars Dölken Jens B Bosse
author_sort	Marco R Rink
title	Concatemeric Broccoli reduces mRNA stability and induces aggregates.
title_short	Concatemeric Broccoli reduces mRNA stability and induces aggregates.
title_full	Concatemeric Broccoli reduces mRNA stability and induces aggregates.
title_fullStr	Concatemeric Broccoli reduces mRNA stability and induces aggregates.
title_full_unstemmed	Concatemeric Broccoli reduces mRNA stability and induces aggregates.
title_sort	concatemeric broccoli reduces mrna stability and induces aggregates.
publisher	Public Library of Science (PLoS)
publishDate	2021
url	https://doaj.org/article/3179583d0e394143b1db375ef4588a2b
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Concatemeric Broccoli reduces mRNA stability and induces aggregates.

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