RNA Detection

attaching a very efficient contact quencher (DN, dinitroaniline) via

a triethylene glycol linker to yield a fluorogenic dye (SR-DN) (see

Fig.3). The fluorescence of SR-DN increases ~100-fold upon

binding to SRB-2 and this dye was utilized to image an RNA of

interest genetically fused to SRB-2 in live bacteria.

1.2 RNA Imaging
Using a Quencher-
Binding Aptamer

Even though fluorophore-binding aptamers are invaluable tools for

in vivo RNA imaging, a new aptamer has to be generated for each

fluorophore with a different structure and a different color, which

hampers the multiplexing potential of fluorophore-binding apta-

mers. Therefore, we decided to generate an RNA aptamer that

binds to dinitroaniline, which was previously discovered as a general

and very efficient contact quencher [17, 18]. With this quencher-

binding aptamer, RNA of interests can be easily labeled with differ-

ent colored fluorophores (seeFig. 1b). It is worth mentioning that

quencher-binding aptamers were previously developed; however,

the quenching mechanism of the fluorescent dyes were based on

PeT [19, 20] or FRET [21] quenching. Due to incomplete

Fig. 2Mfold-predicted secondary structure of (a) SRB-2 aptamer and its binding partner sulforhodamine B
(SR); (b) DNB aptamer and its binding partner dinitroaniline (DN)

Fig. 3Structures of the fluorogenic dyes used in this protocol. Dinitroaniline (DN) and p-nitrobenzylamine (MN)

are the contact quenchers used in this protocol.RG-DNrhodamine green dinitroaniline,TMR-DNtetramethylr-

hodamine dinitroaniline,SR-DNsulforhodamine dinitroaniline,TR-DNTexasRed dinitroaniline,SR-MNsulfor-

hodamine p-nitrobenzylamine

Visualizing RNA with Fluorogenic Aptamers in vivo 291