PT  - JOURNAL ARTICLE
AU  - Marco R. Rink
AU  - Marisa A.P. Baptista
AU  - Thomas Hennig
AU  - Adam W. Whisnant
AU  - Natalia Wolf
AU  - Jürgen Seibel
AU  - Lars Dölken
AU  - Jens B. Bosse
TI  - Concatemeric Broccoli reduces mRNA stability, aggregates and induces p-body formation
AID  - 10.1101/2020.12.07.414458
DP  - 2020 Jan 01
TA  - bioRxiv
PG  - 2020.12.07.414458
4099  - http://biorxiv.org/content/early/2020/12/07/2020.12.07.414458.short
4100  - http://biorxiv.org/content/early/2020/12/07/2020.12.07.414458.full
AB  - 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, a nuclear export defect 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. Overexpression of Broccoli-tagged mRNA led to the formation of p-bodies. However, Broccoli RNAs did not localize to these sites. 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.