2 Development of Fluorogen/Light-Up RNA Aptamer Pairs
As highlighted above, RNA has no intrinsic fluorescence capacity,
but it can acquire fluorescence through specific binding of a fluorogen. Ideally, this fluorogen cofactor should (1) emit no fluorescence in its free state, (2) bind the RNA aptamer with high affinity
(sub-micromolar or even sub-nanomolar K D ), (3) produce strong
fluorescence upon binding, and (4) be photostable. All these properties rely on both partners as demonstrated below. As a consequence, the development of an efficient fluorogen/aptamer pair is a
long and complex multiparametric process in which the fluorogen
should be properly designed and the RNA aptamer subsequently
identified by using a suited selection strategy. This strategy led to
the identification of several tens of pairs covering all the visible
spectrums and displaying ever-improved properties (Table 1).
2.1 Design
of the Fluorogen
Malachite green (MG) was the first fluorogen found to be specifically activated by an RNA aptamer [7]. This was rather an unexpected discovery, since the aptamer was originally developed to
bind MG with the idea of producing reactive radicals aimed at
cleaving RNA in close proximity [11]. The true development of
dedicated fluorogens (most of them are shown in Fig. 2) started
shortly after, leading to a first generation of molecules based on
known nucleic acid-binding dyes such as Hoechst and cyanines like
thiazole orange (TO) and oxazole orange (YO). These molecules
are intrinsically pro-fluorescent. Indeed, Hoechst is an
environment-sensitive molecule that does not emit fluorescence in
Fluorogen
Light-up RNA
aptamer
Fluorescent
complex
Fig. 1 Formation of a fluorogen/light-up aptamer fluorescent complex. In its free
state, the fluorogen (gray) is poorly emissive. However, upon the proper
accommodation into the fluorogen-binding site of a light-up RNA aptamer
(black) the fluorescence capacity of the molecule is restored (green) and a
fluorescent complex is formed
Light-Up RNA Aptamers
75
As highlighted above, RNA has no intrinsic fluorescence capacity,
but it can acquire fluorescence through specific binding of a fluorogen. Ideally, this fluorogen cofactor should (1) emit no fluorescence in its free state, (2) bind the RNA aptamer with high affinity
(sub-micromolar or even sub-nanomolar K D ), (3) produce strong
fluorescence upon binding, and (4) be photostable. All these properties rely on both partners as demonstrated below. As a consequence, the development of an efficient fluorogen/aptamer pair is a
long and complex multiparametric process in which the fluorogen
should be properly designed and the RNA aptamer subsequently
identified by using a suited selection strategy. This strategy led to
the identification of several tens of pairs covering all the visible
spectrums and displaying ever-improved properties (Table 1).
2.1 Design
of the Fluorogen
Malachite green (MG) was the first fluorogen found to be specifically activated by an RNA aptamer [7]. This was rather an unexpected discovery, since the aptamer was originally developed to
bind MG with the idea of producing reactive radicals aimed at
cleaving RNA in close proximity [11]. The true development of
dedicated fluorogens (most of them are shown in Fig. 2) started
shortly after, leading to a first generation of molecules based on
known nucleic acid-binding dyes such as Hoechst and cyanines like
thiazole orange (TO) and oxazole orange (YO). These molecules
are intrinsically pro-fluorescent. Indeed, Hoechst is an
environment-sensitive molecule that does not emit fluorescence in
Fluorogen
Light-up RNA
aptamer
Fluorescent
complex
Fig. 1 Formation of a fluorogen/light-up aptamer fluorescent complex. In its free
state, the fluorogen (gray) is poorly emissive. However, upon the proper
accommodation into the fluorogen-binding site of a light-up RNA aptamer
(black) the fluorescence capacity of the molecule is restored (green) and a
fluorescent complex is formed
Light-Up RNA Aptamers
75