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Aptamer-based RNA imaging & analysis

RNA aptamers are specific RNA sequences that can be detected by cell-permeable small-molecule dyes. Dye fluorescence is significantly enhanced upon complex formation thus aptamers are versatile tags for imaging and functional analysis of coding and non-coding RNA both in vitro and in live cells [1-4]. Attachement of a specific aptamer sequence to a RNA of interest can be performed e.g. by in vitro transcription.


The most commonly used aptamer/dye systems are based on dye derivatives of the GFP chromophore 4-Hydroxybenzlidene (HBI). These 3,5-difluor-HBI dyes (DFHBI, DFHBI-1T and DFHO) show negligible fluorescence in aqueous solutions but light-up upon aptamer complex formation (Tab. 1).

Table 1: Spectroscopic properties of aptamer/dye complexes

DyeLight-up aptamerExc.max/Em.max [nm]
of aptamer/dye complex
ɛmax [M-1cm-1]
of aptamer/dye complex
Spinach 2447/50122000[6]
DFHBI-1TSpinach 2482/50531000[6]

Selected References

[1] Neubacher et al. (2019) RNA Structure and Cellular Applications of Fluorescent Light-Up Aptamers. Angew. Chem. Int. Ed. 58:1266.
[2] Ouellet (2016) RNA Fluorescence with Ligth-Up Aptamers. Front. Chem 4:29.
[3] George et al. (2018) Intracelluar RNA-tracking methods. Open Biol 8:180104.
[4] Dologosheina et al. (2016) Fluorophore Binding RNA aptamers and their application. Wires RNA 7(6):843.
[5] Paige et al. (2011) RNA mimics of green fluorescent protein. Science 333(6042):642.
[6] Song et al. (2014) Plug-and-Play fluorophores extend the Spectral Properties of Spinach. J. Am. Chem. Soc. 136:1198.
[7] Filonov et al. (2014) Broccoli: Rapid Selection of an RNA Mimic of Green Fluorescent Protein by Fluorescence-Based Selection and Directed Evolution. J. Am. Chem. Soc. 136:16299.
[8] Song et al. (2017) Imaging RNA polymerase III transcription using a photostable RNA-fluorophore complex. Nat. Chem. Biol. 13(11):1187.