machinery recently discovered [57]. However, the relaxed constraint exerted on the fluorogen has also two main drawbacks,
i.e., a moderate brightness (moderate QY) and a lack of specificity
of the complex (the aptamer was found to bind and trigger fluorescence not only of OTB-SO3 but also of DIR-Pro and TO1-Biotin).
Yet, both limitations could be overcome by re-exploring the
SELEX-enriched libraries using a functional screening approach
like μIVC. Interestingly, the fluorogen-binding platform of MGA
does not consist of a G-quartet either but instead consists of a base
quadruple (G l G l A l C) that accommodates the fluorogen which is
further capped by a G l C base pair (G8-C28) and surrounded by
several unpaired residues (Fig. 4e) [48]. Despite this a priori tight
accommodation, the complex is still characterized by a low QY
(0.19), but recent experiments showed that this value could be
significantly increased using in vitro functional screening [37].
Among the different structurally characterized light-up RNA
aptamers, DFHBI-(1 T)-binding Spinach aptamers (Spinach [18],
Spinach2 [58], iSpinach [42]) possess the most elaborated
fluorogen-binding pocket (Fig. 4f). Indeed, all the solved crystal
structures showed that the fluorogen-binding platform is made of a
G-quartet and that DFHBI(-1T) is accommodated between this
platform and a base triple, while specific contacts are established
with a side guanine (G31) [49–51]. Exploiting the crystal structure
allowed to truncate Spinach to a much shorter aptamer called BabySpinach that preserves the properties of the parental molecule
[50]. In addition to the Spinach family aptamers, it is very likely
that the same DFHBI(-1T)-binding pocket is shared by Broccoli
[35]. Indeed, even though no crystal structure has been solved for
this aptamer, the close sequence proximity with Spinach strongly
suggests that both molecules adopt the same folding [59–
61]. However, despite the tight apparent accommodation of the
fluorogen, all the known DFHBI(-1T)/aptamer complexes suffer
from a very short fluorescence half-life (less than 1 s) [23]. This is
likely due to the loose constraint applied to the imidazolinone
moiety that stays free to eliminate part of the excitation energy
through rapid photoisomerization.
The precursor of Broccoli was recently subjected to rounds of
directed evolution and converted into Orange Broccoli and Red
Broccoli, two aptamers able to light up DFHO, a red fluorescent
protein-mimicking fluorogen, at different wavelengths [21]. Comparing the sequences of the three (the original, Orange and Red)
Broccolis as well as considering their putative Spinach-like DFHBI
(-1T)-binding pocket allowed to identify a single nucleotide
responsible for the Broccoli spectral tuning [60]. While this feature
makes Broccoli an interesting precursor for the development of
multicolor tags, this aptamer family is still limited by the poor
photostability and the limited brightness of the complex they
form with their fluorogens [21]. However, significant gain in
86
Michael Ryckelynck
i.e., a moderate brightness (moderate QY) and a lack of specificity
of the complex (the aptamer was found to bind and trigger fluorescence not only of OTB-SO3 but also of DIR-Pro and TO1-Biotin).
Yet, both limitations could be overcome by re-exploring the
SELEX-enriched libraries using a functional screening approach
like μIVC. Interestingly, the fluorogen-binding platform of MGA
does not consist of a G-quartet either but instead consists of a base
quadruple (G l G l A l C) that accommodates the fluorogen which is
further capped by a G l C base pair (G8-C28) and surrounded by
several unpaired residues (Fig. 4e) [48]. Despite this a priori tight
accommodation, the complex is still characterized by a low QY
(0.19), but recent experiments showed that this value could be
significantly increased using in vitro functional screening [37].
Among the different structurally characterized light-up RNA
aptamers, DFHBI-(1 T)-binding Spinach aptamers (Spinach [18],
Spinach2 [58], iSpinach [42]) possess the most elaborated
fluorogen-binding pocket (Fig. 4f). Indeed, all the solved crystal
structures showed that the fluorogen-binding platform is made of a
G-quartet and that DFHBI(-1T) is accommodated between this
platform and a base triple, while specific contacts are established
with a side guanine (G31) [49–51]. Exploiting the crystal structure
allowed to truncate Spinach to a much shorter aptamer called BabySpinach that preserves the properties of the parental molecule
[50]. In addition to the Spinach family aptamers, it is very likely
that the same DFHBI(-1T)-binding pocket is shared by Broccoli
[35]. Indeed, even though no crystal structure has been solved for
this aptamer, the close sequence proximity with Spinach strongly
suggests that both molecules adopt the same folding [59–
61]. However, despite the tight apparent accommodation of the
fluorogen, all the known DFHBI(-1T)/aptamer complexes suffer
from a very short fluorescence half-life (less than 1 s) [23]. This is
likely due to the loose constraint applied to the imidazolinone
moiety that stays free to eliminate part of the excitation energy
through rapid photoisomerization.
The precursor of Broccoli was recently subjected to rounds of
directed evolution and converted into Orange Broccoli and Red
Broccoli, two aptamers able to light up DFHO, a red fluorescent
protein-mimicking fluorogen, at different wavelengths [21]. Comparing the sequences of the three (the original, Orange and Red)
Broccolis as well as considering their putative Spinach-like DFHBI
(-1T)-binding pocket allowed to identify a single nucleotide
responsible for the Broccoli spectral tuning [60]. While this feature
makes Broccoli an interesting precursor for the development of
multicolor tags, this aptamer family is still limited by the poor
photostability and the limited brightness of the complex they
form with their fluorogens [21]. However, significant gain in
86
Michael Ryckelynck