[27, 28]. The beauty of this approach is the possibility of generating a variety of dyes with different colors. Indeed, grafting
DN-PEG to various dyes with xanthene-like cores (e.g., RG-DN
and TMR-DN) allowed generating a rainbow of fluorogens [29]
and led recently to the development of the very bright silicon
rhodamine-DN fluorogen [30]. Interestingly, exploiting the internal spirocyclization (a spontaneous intramolecular cyclization event
abrogating the fluorescence of the molecule but reversible by the
binding to an aptamer) capacity of the silicon rhodamine even made
the addition of the quencher moiety (i.e., SR linker) dispensable,
yet at the expense of a lower fluorescence turn-on, thus leading to a
lower contrast [30]. Alternative designs in which DN was
exchanged for FRET quenchers like Black Hole Quencher 1 (e.g.,
Cy3-BHQ1) [31] and, more recently, cobalamin (e.g., Cbl-Cy5
and Cbl-Atto590) [32] were also described and enabled the development of multicolor imaging platforms. The use of cobalamin
(Cbl) as a quencher is a very clever strategy as it exploits a natural
compound for which several natural and specifically interacting
RNA aptamer sequences were already identified in bacterial riboswitches. Fortunately, this interaction with Cbl is efficient enough to
abrogate the quenching of the dye, making the de novo isolation of
a dedicated aptamer dispensable.
2.2 Development
and Properties
of Artificial Light-Up
RNA Aptamers
With the exception of Cbl-binding RNAs introduced above, no
other natural light-up RNA aptamer has been described yet. While
the absence of such molecule in cells limits the risk of unwanted
background fluorescence resulting from a cross activation by
endogenous sequences, it also requires the development of an
artificial aptamer. Over the past decade, several strategies have
been proposed to isolate and optimize light-up RNA aptamers.
2.2.1 Aptamer Isolation
Strategies
Traditionally, artificial aptamers are obtained using an in vitro selection technology known as SELEX (Systematic Evolution of
Ligands by EXponential enrichment) introduced in the early
1990s [33, 34]. In this approach, RNA (or DNA) molecules
contained in large libraries made of 10
14–15 different sequences
are challenged to interact with an immobilized target, here, a
fluorogen (Fig. 3a). Upon stringent washes, molecules displaying
sufficient affinity for the target are recovered and the resulting
enriched library is used to prime another round of selection. As a
consequence, aptamers isolated by SELEX are likely to be specific
and high-affinity binders. Consistent with this assumption, most of
the aptamers recognize their fluorogen with nanomolar affinity
(Table 1). The best systems described so far in this regard are the
TO1-Biotin/Mango [14], DMHBO+/Chili [22], and TMR-DN/
SRB2 systems showing K D values of 3 nM, 12 nM, and 35 nM,
respectively [29].
Light-Up RNA Aptamers
79
DN-PEG to various dyes with xanthene-like cores (e.g., RG-DN
and TMR-DN) allowed generating a rainbow of fluorogens [29]
and led recently to the development of the very bright silicon
rhodamine-DN fluorogen [30]. Interestingly, exploiting the internal spirocyclization (a spontaneous intramolecular cyclization event
abrogating the fluorescence of the molecule but reversible by the
binding to an aptamer) capacity of the silicon rhodamine even made
the addition of the quencher moiety (i.e., SR linker) dispensable,
yet at the expense of a lower fluorescence turn-on, thus leading to a
lower contrast [30]. Alternative designs in which DN was
exchanged for FRET quenchers like Black Hole Quencher 1 (e.g.,
Cy3-BHQ1) [31] and, more recently, cobalamin (e.g., Cbl-Cy5
and Cbl-Atto590) [32] were also described and enabled the development of multicolor imaging platforms. The use of cobalamin
(Cbl) as a quencher is a very clever strategy as it exploits a natural
compound for which several natural and specifically interacting
RNA aptamer sequences were already identified in bacterial riboswitches. Fortunately, this interaction with Cbl is efficient enough to
abrogate the quenching of the dye, making the de novo isolation of
a dedicated aptamer dispensable.
2.2 Development
and Properties
of Artificial Light-Up
RNA Aptamers
With the exception of Cbl-binding RNAs introduced above, no
other natural light-up RNA aptamer has been described yet. While
the absence of such molecule in cells limits the risk of unwanted
background fluorescence resulting from a cross activation by
endogenous sequences, it also requires the development of an
artificial aptamer. Over the past decade, several strategies have
been proposed to isolate and optimize light-up RNA aptamers.
2.2.1 Aptamer Isolation
Strategies
Traditionally, artificial aptamers are obtained using an in vitro selection technology known as SELEX (Systematic Evolution of
Ligands by EXponential enrichment) introduced in the early
1990s [33, 34]. In this approach, RNA (or DNA) molecules
contained in large libraries made of 10
14–15 different sequences
are challenged to interact with an immobilized target, here, a
fluorogen (Fig. 3a). Upon stringent washes, molecules displaying
sufficient affinity for the target are recovered and the resulting
enriched library is used to prime another round of selection. As a
consequence, aptamers isolated by SELEX are likely to be specific
and high-affinity binders. Consistent with this assumption, most of
the aptamers recognize their fluorogen with nanomolar affinity
(Table 1). The best systems described so far in this regard are the
TO1-Biotin/Mango [14], DMHBO+/Chili [22], and TMR-DN/
SRB2 systems showing K D values of 3 nM, 12 nM, and 35 nM,
respectively [29].
Light-Up RNA Aptamers
79