RNA Detection

1.1 RNA Imaging
Using a Fluorophore-
Binding Aptamer

Previously, several short RNA sequences (aptamers) which bind to

specific fluorogenic dyes [6–8] or fluorophores such as fluorescein

[9], sulforhodamine B [9], and tetramethylrhodamine [10] have

been developed by using Systematic Evolution of Ligands by EXpo-

nential enrichment (SELEX) [11]. However, they were not utilized

for in vivo RNA imaging applications, presumably due to low

affinity, toxicity of the dyes or high background fluorescence in

cells. Lately, an aptamer, named Spinach, that binds to 3,5-

difluoro-4-hydroxybenzylidine imidazolinone (DFHBI, a mimic

of the GFP chromophore) has been developed and utilized for

in vivo RNA imaging [12]. Spinach was genetically encoded to

the target RNAs, which become fluorescent upon binding of the

fluorogenic dye to the aptamer. The first generation Spinach apta-

mer and DFHBI dye were further coevolved to create more stable,

smaller and brighter aptamers [13–15]. In addition to Spinach and

its derivatives, recently an aptamer, named Mango, with an

extremely high affinity to thiazole orange was discovered and

appeared to be a promising tool for in vivo RNA imaging [16].

We recently developed a new method combining contact-

quenched (seeNote 1) fluorogenic dyes and a fluorophore-binding

aptamer to image RNA in live bacterial cells (seeFig. 1a)[17]. This

method was based on noncovalent interactions between a

fluorophore-binding aptamer (SRB-2, sulforhodamine B binding

aptamer) and a fluorophore (SR, sulforhodamine B) (seeFig. 2a). In

this approach, the fluorescence of SR was diminished drastically by

Fig. 1Principles of the RNA imaging method based on fluorophore- and quencher-binding aptamers. The
contact-quenched fluorophore–quencher conjugates (OFF) light up upon binding to (a) a fluorophore binding
aptamer, or (b) a quencher-binding aptamer (ON). RNA of interest can be fused to one of the fluorescence
enhancing aptamers and imaged in the presence of the fluorophore–quencher conjugate. F denotes any
fluorophore and Q denotes a contact quencher. Images were reproduced from [22] with permission from
Oxford University Press

290 Murat Sunbul et al.