approaches allow detecting nucleic acids with great specificity and
sensitivity, they also require cells to be fixed and permeabilized,
which compromises live-cell imaging and leads to a significant loss
of information on the dynamics of the biological system. Other
probes and nanoparticles such as molecular beacons and nanoflares
have been proposed as an alternative to fluorescent ISH (FISH) but
these probes face cell entry and toxicity issues [4].
A first breakthrough in live-cell RNA imaging came with the
introduction of the “RBP-FP” methods pioneered by Bertrand
et al. in late 1990s with the so-called MS2-GFP system [5]. These
approaches exploit the capacity of some RNA-binding proteins
(RBP) to specifically recognize short RNA motifs herein used as
RNA tags. The RNA to image is then expressed in cells in fusion
(usually in the 3
0 untranslated region of mRNAs) with an array of
RNA tags (several tens of motifs tandemly repeated). In addition, a
construct coding for the RBP specific to the RNA tag and fused
with a fluorescent protein (FP), such as the green fluorescent
protein (GFP), is also expressed in the same cell. As a consequence,
upon synthesis, the target mRNA is rapidly decorated with tens of
GFPs turning it into a highly fluorescent object that can be imaged
with single-molecule resolution [6]. Yet, whereas this technology
allowed collecting highly valuable data on mRNA synthesis, addressing, and distribution in the cell, it also suffers significant drawbacks
linked to the large size of the tag array (up to 32 repeats of the RNA
motif), especially when decorated by the RBP-FP, as well as the
background coming from the constitutively expressed RBP-FP,
though adding a nuclear localization sequence can be used to
confine unbound RBP-FP into the nucleus. These features preclude the use of this technology for imaging small, yet highly
relevant, noncoding RNAs but also its application to organisms
deprived of internal compartments (e.g., bacteria). Yet, these
major limitations can be overcome by reducing the length of
RNA tag arrays and by exchanging the bulky fluorescent RBP-FP
for a smaller pro-fluorescent molecule.
Conversely to proteins, no naturally fluorescent RNA has been
discovered yet, making it necessary to use a pro-fluorescent cofactor. In 2003 the group of Roger Tsien reported that the so-called
MGA RNA aptamer binds specifically to malachite green and other
triphenylmethane dyes and that this interaction activates their fluorescence [7], making these dyes fluorogenic (Fig. 1). Since this
pioneering work, a large variety of such dyes (“fluorogens”) and
RNA aptamers (“light-up aptamers”) forming specific fluorogen/
light-up aptamer pairs have been developed and they are still subject of a very active field of research today [8–10]. This chapter aims
at reviewing and discussing the development of the main fluorogen/light-up aptamer pairs that are currently available. In addition,
some of their applications in live-cell RNA imaging as well as in
biosensing and nanotechnology are presented.
74
Michael Ryckelynck
sensitivity, they also require cells to be fixed and permeabilized,
which compromises live-cell imaging and leads to a significant loss
of information on the dynamics of the biological system. Other
probes and nanoparticles such as molecular beacons and nanoflares
have been proposed as an alternative to fluorescent ISH (FISH) but
these probes face cell entry and toxicity issues [4].
A first breakthrough in live-cell RNA imaging came with the
introduction of the “RBP-FP” methods pioneered by Bertrand
et al. in late 1990s with the so-called MS2-GFP system [5]. These
approaches exploit the capacity of some RNA-binding proteins
(RBP) to specifically recognize short RNA motifs herein used as
RNA tags. The RNA to image is then expressed in cells in fusion
(usually in the 3
0 untranslated region of mRNAs) with an array of
RNA tags (several tens of motifs tandemly repeated). In addition, a
construct coding for the RBP specific to the RNA tag and fused
with a fluorescent protein (FP), such as the green fluorescent
protein (GFP), is also expressed in the same cell. As a consequence,
upon synthesis, the target mRNA is rapidly decorated with tens of
GFPs turning it into a highly fluorescent object that can be imaged
with single-molecule resolution [6]. Yet, whereas this technology
allowed collecting highly valuable data on mRNA synthesis, addressing, and distribution in the cell, it also suffers significant drawbacks
linked to the large size of the tag array (up to 32 repeats of the RNA
motif), especially when decorated by the RBP-FP, as well as the
background coming from the constitutively expressed RBP-FP,
though adding a nuclear localization sequence can be used to
confine unbound RBP-FP into the nucleus. These features preclude the use of this technology for imaging small, yet highly
relevant, noncoding RNAs but also its application to organisms
deprived of internal compartments (e.g., bacteria). Yet, these
major limitations can be overcome by reducing the length of
RNA tag arrays and by exchanging the bulky fluorescent RBP-FP
for a smaller pro-fluorescent molecule.
Conversely to proteins, no naturally fluorescent RNA has been
discovered yet, making it necessary to use a pro-fluorescent cofactor. In 2003 the group of Roger Tsien reported that the so-called
MGA RNA aptamer binds specifically to malachite green and other
triphenylmethane dyes and that this interaction activates their fluorescence [7], making these dyes fluorogenic (Fig. 1). Since this
pioneering work, a large variety of such dyes (“fluorogens”) and
RNA aptamers (“light-up aptamers”) forming specific fluorogen/
light-up aptamer pairs have been developed and they are still subject of a very active field of research today [8–10]. This chapter aims
at reviewing and discussing the development of the main fluorogen/light-up aptamer pairs that are currently available. In addition,
some of their applications in live-cell RNA imaging as well as in
biosensing and nanotechnology are presented.
74
Michael Ryckelynck