Abstract
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.
Highlights
RNA aptamers are short RNA sequences that exert specific binding abilities to a given biological structure or small molecule
The coding sequence (CDS) of the fluorescent protein mCherry was inserted upstream of the Broccoli tandem arrays to monitor their effects on protein expression
Fluorogenic RNA aptamers have the potential of surpassing the state-of-the-art real-time RNA imaging technology
Summary
RNA aptamers are short RNA sequences that exert specific binding abilities to a given biological structure or small molecule. Fluorogenic RNA aptamers in this regard are characterized by their ability to bind a small molecule and greatly enhance its fluorescence potential. This has been first reported with an aptamer called Spinach [1] (the name due to its green fluorescence) and been further expanded over the recent years with other aptamers emerging, such as the RNA Mangos [2], Broccoli [3] or Corn [4]. The most prevalent RNA aptamers are RNA mimics of GFP, termed as such by the very similar peak excitation and emission wavelengths These aptamers consist of binding a GFP mimicking small molecule.
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