identified by employing high-throughput screening approaches
such as the μIVC-based procedure we recently described
[126]. Alternative design strategies have been proposed using natural riboswitches and circularly permutated molecules. However,
since these sensors and their development are out of the scope of
this chapter, the reader is redirected to some excellent recent
reviews on the topic [127–129]. While for a long time the use of
these sensors in mammalian cells was challenged by the short halflife of small RNAs in these cells, the recent development of the
Tornado technology allows to express these sensors as highly stable
circular RNAs [68].
Exploiting RNA nanotechnology also led to the designing of a
construct comprising Spinach and Mango aptamers positioned in
such a way that FRET can occur between the fluorogens (respectively, DFHBI-1T and YO3) bound to each aptamer. Moreover,
inserting a metabolite-sensing aptamer into the construct allows to
couple the FRET signal to a metabolite-sensing event [15]. Interestingly, metabolite biosensors can also be used as extracellular
probes in screening experiments aiming at identifying microbes
with improved capacity to synthesize and secrete a target
metabolite [130].
Last but least, light-up aptamers can easily be converted into
ion sensors. The two most prominent examples are silver and lead
sensors. Indeed, simply converting a Watson-Crick base pair of
Broccoli into a C l C mismatch allowed to transiently abrogate the
fluorogen-binding capacity of the aptamer. However, in the presence of silver ions, a C-Ag
+ -C metallo base pair forms and restores
aptamer capacity to bind and trigger DFHBI-1T fluorescence
[131]. Expressing this sensor in living bacteria permitted to titrate
the amount of silver that can actually enter and accumulate into the
cells. The second example is lead detection. Indeed, it was found
that the G-quadruplex structure contained in Spinach can strongly
bind and get stabilized by Pb
2+ ions, making Spinach an excellent
sensor able to specifically detect as few as 6 nM Pb
2+
, a concentration far below the maximum permissible concentration (72 nM)
[132]. Moreover, it was recently found that a 2
0 -fluorinated version
of the sensor may offer even higher performance by protecting the
sensor from RNase action and by enhancing the association with
Pb
2+ ions [133]. Interestingly, exploiting this Pb
2+ binding capacity
of Spinach enabled the development of a fluorescence-free liquid
crystal sensor able to detect Pb
2+ ions with similar selectivity and
sensitivity [134], demonstrating how properties of light-up aptamers can even be used without exploiting their fluorescence capacity. In the same line, the very high affinity of Mango for TO1-Biotin
can be exploited in experiments aiming to specifically isolate target
RNAs and bound molecules by affinity capture [135].
94
Michael Ryckelynck
such as the μIVC-based procedure we recently described
[126]. Alternative design strategies have been proposed using natural riboswitches and circularly permutated molecules. However,
since these sensors and their development are out of the scope of
this chapter, the reader is redirected to some excellent recent
reviews on the topic [127–129]. While for a long time the use of
these sensors in mammalian cells was challenged by the short halflife of small RNAs in these cells, the recent development of the
Tornado technology allows to express these sensors as highly stable
circular RNAs [68].
Exploiting RNA nanotechnology also led to the designing of a
construct comprising Spinach and Mango aptamers positioned in
such a way that FRET can occur between the fluorogens (respectively, DFHBI-1T and YO3) bound to each aptamer. Moreover,
inserting a metabolite-sensing aptamer into the construct allows to
couple the FRET signal to a metabolite-sensing event [15]. Interestingly, metabolite biosensors can also be used as extracellular
probes in screening experiments aiming at identifying microbes
with improved capacity to synthesize and secrete a target
metabolite [130].
Last but least, light-up aptamers can easily be converted into
ion sensors. The two most prominent examples are silver and lead
sensors. Indeed, simply converting a Watson-Crick base pair of
Broccoli into a C l C mismatch allowed to transiently abrogate the
fluorogen-binding capacity of the aptamer. However, in the presence of silver ions, a C-Ag
+ -C metallo base pair forms and restores
aptamer capacity to bind and trigger DFHBI-1T fluorescence
[131]. Expressing this sensor in living bacteria permitted to titrate
the amount of silver that can actually enter and accumulate into the
cells. The second example is lead detection. Indeed, it was found
that the G-quadruplex structure contained in Spinach can strongly
bind and get stabilized by Pb
2+ ions, making Spinach an excellent
sensor able to specifically detect as few as 6 nM Pb
2+
, a concentration far below the maximum permissible concentration (72 nM)
[132]. Moreover, it was recently found that a 2
0 -fluorinated version
of the sensor may offer even higher performance by protecting the
sensor from RNase action and by enhancing the association with
Pb
2+ ions [133]. Interestingly, exploiting this Pb
2+ binding capacity
of Spinach enabled the development of a fluorescence-free liquid
crystal sensor able to detect Pb
2+ ions with similar selectivity and
sensitivity [134], demonstrating how properties of light-up aptamers can even be used without exploiting their fluorescence capacity. In the same line, the very high affinity of Mango for TO1-Biotin
can be exploited in experiments aiming to specifically isolate target
RNAs and bound molecules by affinity capture [135].
94
Michael Ryckelynck