its free state, but becomes highly fluorescent when bound to DNA
[9]. On their side, TO and YO have the capacity to eliminate an
excitation energy via intramolecular movements in their free state.
However, binding to nucleic acids hinders intramolecular movements and the excitation energy is then eliminated by fluorescence
emission. To serve as specific fluorogen, the nonspecific binding
capacity of these dyes was strongly attenuated by introducing
blocking chemical groups, leading to compounds with a potential
to interact with nucleic acids but becoming fluorescent only upon
binding to a specific sequence. This first set of fluorogens encompasses molecules like Hoechst derivative 1c [12, 13], TO1-Biotin
[14], TO3-Biotin [14], YO3 [15], DIR [16], DIR-pro [17], and
OTB-SO3 [17] (Table 1). In addition to these repurposed dyes,
bio-inspired fluorogens (also behaving as molecular rotors) were
designed by mimicking fluorophores found in fluorescent proteins.
In doing so, the group of Samie Jaffrey introduced DFHBI
(3,5-difluoro-4-hydroxybenzylidene imidazolinone) as the first
GFP-mimicking fluorogen [18] together with DFHBI-1T, a
brighter derivative [19]. Moreover, since green fluorescence may
also originate from cell autofluorescence, several groups later developed red-emitting FP-mimicking fluorogens [20–22]. Interestingly, protonated forms of these FP-mimicking fluorogens (e.g.,
DMHBI
+
, DMHBI-Imi, and DMHBO
+
) represent large stokeshift fluorogens, which are particularly attractive for Fo ¨rster resonance energy transfer (FRET) applications [22]. This first generation of fluorogens already covered most of the visible spectrum
(Fig. 2) and allowed a real breakthrough to begin in RNA imaging
technologies (see below). However, these dyes suffer from a limited
brightness (Table 1) and, in most of the cases, a low photostability—the most extreme case being encountered with the DFHBI
and DFHBI-1 T (from now summarized as DFHBI(À1 T)) fluorogens that, when associated with their specific aptamer, produce
fluorescence for less than a second before getting photobleached
[23, 24].
The abovementioned limitations encouraged the development
of a second set of fluorogens based on organic dyes known to be
both bright and photostable (e.g., rhodamines, Atto, and Alexa).
Yet, these molecules first needed to be converted into reversible
non-emissive species (Fig. 2). To do so, the molecule is usually
conjugated with a quenching moiety that prevents fluorescence
emission by either contact quenching, FRET, or electron transfer
[25]. For instance, direct addition of an aniline to an amino group
of a sulforhodamine core led to ASR, a dye quenched by an electron
transfer mechanism revertible upon aptamer binding [26]. Sulforhodamine B (SRB) can also be conjugated to dinitroaniline
(DN) via a short polyethylene glycol (PEG) linker introduced at
the level of a sulfone group, yielding a fluorogen (i.e., SR-DN)
rendered non-emissive by a contact quenching phenomenon,
78
Michael Ryckelynck
[9]. On their side, TO and YO have the capacity to eliminate an
excitation energy via intramolecular movements in their free state.
However, binding to nucleic acids hinders intramolecular movements and the excitation energy is then eliminated by fluorescence
emission. To serve as specific fluorogen, the nonspecific binding
capacity of these dyes was strongly attenuated by introducing
blocking chemical groups, leading to compounds with a potential
to interact with nucleic acids but becoming fluorescent only upon
binding to a specific sequence. This first set of fluorogens encompasses molecules like Hoechst derivative 1c [12, 13], TO1-Biotin
[14], TO3-Biotin [14], YO3 [15], DIR [16], DIR-pro [17], and
OTB-SO3 [17] (Table 1). In addition to these repurposed dyes,
bio-inspired fluorogens (also behaving as molecular rotors) were
designed by mimicking fluorophores found in fluorescent proteins.
In doing so, the group of Samie Jaffrey introduced DFHBI
(3,5-difluoro-4-hydroxybenzylidene imidazolinone) as the first
GFP-mimicking fluorogen [18] together with DFHBI-1T, a
brighter derivative [19]. Moreover, since green fluorescence may
also originate from cell autofluorescence, several groups later developed red-emitting FP-mimicking fluorogens [20–22]. Interestingly, protonated forms of these FP-mimicking fluorogens (e.g.,
DMHBI
+
, DMHBI-Imi, and DMHBO
+
) represent large stokeshift fluorogens, which are particularly attractive for Fo ¨rster resonance energy transfer (FRET) applications [22]. This first generation of fluorogens already covered most of the visible spectrum
(Fig. 2) and allowed a real breakthrough to begin in RNA imaging
technologies (see below). However, these dyes suffer from a limited
brightness (Table 1) and, in most of the cases, a low photostability—the most extreme case being encountered with the DFHBI
and DFHBI-1 T (from now summarized as DFHBI(À1 T)) fluorogens that, when associated with their specific aptamer, produce
fluorescence for less than a second before getting photobleached
[23, 24].
The abovementioned limitations encouraged the development
of a second set of fluorogens based on organic dyes known to be
both bright and photostable (e.g., rhodamines, Atto, and Alexa).
Yet, these molecules first needed to be converted into reversible
non-emissive species (Fig. 2). To do so, the molecule is usually
conjugated with a quenching moiety that prevents fluorescence
emission by either contact quenching, FRET, or electron transfer
[25]. For instance, direct addition of an aniline to an amino group
of a sulforhodamine core led to ASR, a dye quenched by an electron
transfer mechanism revertible upon aptamer binding [26]. Sulforhodamine B (SRB) can also be conjugated to dinitroaniline
(DN) via a short polyethylene glycol (PEG) linker introduced at
the level of a sulfone group, yielding a fluorogen (i.e., SR-DN)
rendered non-emissive by a contact quenching phenomenon,
78
Michael Ryckelynck