the environment of the dye (Fig. 4b). Moreover, while held in
planar conformation, the fluorogen is also slightly more exposed
to the solvent. Taken together, these observations explained the
increased affinity of Mango-II for TO1-Biotin (better accommodation of the fluorogen) while the brightness of the complex
remained unchanged (no advantageous modification of the fluorogen environment). Mango-I and -II are both able to trigger
TO1-Biotin fluorescence and to a lower extent that of the related,
red-emitting analog TO3-Biotin. However, guided by the crystal
structure, we identified a point mutant of Mango-II (Mango-II
(A22U)) endowed with a better capacity to discriminate both
fluorogens [54]. Interestingly, and as expected from the significant
sequence differences, Mango-III displays a rather different structural organization [55]. Indeed, whereas the RNA still possesses a
G-quadruplex of which the top quartet forms the fluorogenbinding platform, the aptamer has a much more compact and
robust structure in which almost every residue is involved in hydrogen bonding. Moreover, and conversely to other Mangos, the
G-quartet platform of Mango-III is not surmounted by unpaired
nucleobases but is rather capped by a A-U base pair (A10-U17) that
sandwiches the fluorogen and is stabilized by an intercalating U
(U12) residue (Fig. 4c). As a direct consequence of this tight
accommodation of TO1-Biotin between the G-quadruplex platform and the apical A-U base-pair, the QY of TO1-Biotin/MangoIII is almost four times higher than that of other Mango aptamers
without significant change in affinity (Table 1). Moreover, we
found out that Mango-III almost completely lost the capacity to
trigger TO3-Biotin fluorescence, making this aptamer one of the
most specific aptamer known to date. As before, guided by the
crystal structure, we identified ten residues that could potentially
be suboptimal [55]. We prepared a mutant library in which these
ten positions were randomized, and we screened it for improved
variants using μIVC. Excitingly, this allowed us to identify iMangoIII, an aptamer forming an even brighter complex with TO1-Biotin
and with preserved affinity (Table 1). The overall structure of
iMango-III is identical to that of Mango-III [55, 56], the main
difference being the exchange of the A-U apical closing base pair
for a U l U pair that was identified as being responsible for the
increase in brightness of the complex.
Recently, the crystal structure of the DIR2s aptamer was solved
in complex with the fluorogen OTB-SO3 [47]. Interestingly, the
fluorogen-binding platform of DIR2s consists only of a base triple,
and the fluorogen is capped by a single adenine (A40, Fig. 4d).
Moreover, the fluorogen establishes a specific interaction with a
guanine (G39) of the platform via its sulfone moiety. Interestingly,
it was proposed that the absence of G-quadruplex should render
this aptamer less sensitive to the cellular G-quadruplex unfolding
Light-Up RNA Aptamers
85
planar conformation, the fluorogen is also slightly more exposed
to the solvent. Taken together, these observations explained the
increased affinity of Mango-II for TO1-Biotin (better accommodation of the fluorogen) while the brightness of the complex
remained unchanged (no advantageous modification of the fluorogen environment). Mango-I and -II are both able to trigger
TO1-Biotin fluorescence and to a lower extent that of the related,
red-emitting analog TO3-Biotin. However, guided by the crystal
structure, we identified a point mutant of Mango-II (Mango-II
(A22U)) endowed with a better capacity to discriminate both
fluorogens [54]. Interestingly, and as expected from the significant
sequence differences, Mango-III displays a rather different structural organization [55]. Indeed, whereas the RNA still possesses a
G-quadruplex of which the top quartet forms the fluorogenbinding platform, the aptamer has a much more compact and
robust structure in which almost every residue is involved in hydrogen bonding. Moreover, and conversely to other Mangos, the
G-quartet platform of Mango-III is not surmounted by unpaired
nucleobases but is rather capped by a A-U base pair (A10-U17) that
sandwiches the fluorogen and is stabilized by an intercalating U
(U12) residue (Fig. 4c). As a direct consequence of this tight
accommodation of TO1-Biotin between the G-quadruplex platform and the apical A-U base-pair, the QY of TO1-Biotin/MangoIII is almost four times higher than that of other Mango aptamers
without significant change in affinity (Table 1). Moreover, we
found out that Mango-III almost completely lost the capacity to
trigger TO3-Biotin fluorescence, making this aptamer one of the
most specific aptamer known to date. As before, guided by the
crystal structure, we identified ten residues that could potentially
be suboptimal [55]. We prepared a mutant library in which these
ten positions were randomized, and we screened it for improved
variants using μIVC. Excitingly, this allowed us to identify iMangoIII, an aptamer forming an even brighter complex with TO1-Biotin
and with preserved affinity (Table 1). The overall structure of
iMango-III is identical to that of Mango-III [55, 56], the main
difference being the exchange of the A-U apical closing base pair
for a U l U pair that was identified as being responsible for the
increase in brightness of the complex.
Recently, the crystal structure of the DIR2s aptamer was solved
in complex with the fluorogen OTB-SO3 [47]. Interestingly, the
fluorogen-binding platform of DIR2s consists only of a base triple,
and the fluorogen is capped by a single adenine (A40, Fig. 4d).
Moreover, the fluorogen establishes a specific interaction with a
guanine (G39) of the platform via its sulfone moiety. Interestingly,
it was proposed that the absence of G-quadruplex should render
this aptamer less sensitive to the cellular G-quadruplex unfolding
Light-Up RNA Aptamers
85