engineering a domain of the SRB2 aptamer into a molecular beacon
[77]. In this construct, the successful interaction between the
sensing loop of the probe and the target miR leads to structural
rearrangements activating SRB2, which in turn can bind and trigger
the bright SR-DN fluorogen. More recently, a new concept in
which the probe is made of an L-enantiomer of Mango-III supplied
from outside the cell has been introduced [78]. L-enantiomers are
naturally resistant to nuclease, making these probes extremely longlasting in challenging media (e.g., extracellular space). Furthermore, coupling the probe to a cholesterol molecule and using a
clever LNA-based blocker allowed cell entry of the probe and its
activation by cellular miR (natural D-enantiomer).
Finally, a light-up aptamer can be converted into a probe acting
in trans by splitting it and appending each half to a sequence
complementary to the ROI (Fig. 5e). Therefore, a functional aptamer is reconstituted only in the presence of the target RNA that acts
as a scaffold driving aptamer assembly. Split aptamer-based methodologies were shown to offer a very high specificity [79–81]. The
recent application of this concept in live cells allowed mRNA to be
detected from genetically encoded Spinach and Broccoli-derived
probes with a decent sensitivity (minimum detectable concentration of 50–100 nM) in living cells [82]. Substantial gain in sensitivity was further obtained by combining the use of a split Broccoli
aptamer and a catalytic hairpin assembly amplification circuit
(a molecular amplification circuit derived from RNA nanotechnology). The resulting CHARGE technology (Fig. 5f) allows detecting the presence of as few as 2.5 nM of target RNAs in live cells in a
digital manner (i.e., informs on the presence or absence of the
target) [83]. Last but not least, split aptamers can also be expressed
in cells and used to detect RNA-RNA interactions [84].
3.4 In Vitro Detection
of RNA
The key implication of miRs in various diseases and disorders has
stimulated the development of sensitive in vitro detection technologies for diagnostic applications. In this view, light-up RNA aptamers offer the great advantage of allowing to design cheap, labelfree, and sensitive assays. Following the same concepts as for livecell RNA imaging, the first generation of in vitro RNA sensors
exploited transiently destabilized aptamers (e.g., PANDAN technology [75]) and split aptamer [81] strategies. However, the
micromolar to high nanomolar sensitivity of these technologies
was insufficient to detect low-abundant RNAs suggesting that an
amplification step may be necessary. Several RNA-based molecular
amplification circuits, including CHARGE, have been developed
for in vitro applications [83, 85, 86] and could be used to increase
detection sensitivity. However, an elegant alternative consisting of
using the amplification capacity of the in vitro transcription reaction
has recently been proposed and revealed to be extremely efficient
for the sensitive detection of nucleic acids [87, 88] and other classes
Light-Up RNA Aptamers
91
[77]. In this construct, the successful interaction between the
sensing loop of the probe and the target miR leads to structural
rearrangements activating SRB2, which in turn can bind and trigger
the bright SR-DN fluorogen. More recently, a new concept in
which the probe is made of an L-enantiomer of Mango-III supplied
from outside the cell has been introduced [78]. L-enantiomers are
naturally resistant to nuclease, making these probes extremely longlasting in challenging media (e.g., extracellular space). Furthermore, coupling the probe to a cholesterol molecule and using a
clever LNA-based blocker allowed cell entry of the probe and its
activation by cellular miR (natural D-enantiomer).
Finally, a light-up aptamer can be converted into a probe acting
in trans by splitting it and appending each half to a sequence
complementary to the ROI (Fig. 5e). Therefore, a functional aptamer is reconstituted only in the presence of the target RNA that acts
as a scaffold driving aptamer assembly. Split aptamer-based methodologies were shown to offer a very high specificity [79–81]. The
recent application of this concept in live cells allowed mRNA to be
detected from genetically encoded Spinach and Broccoli-derived
probes with a decent sensitivity (minimum detectable concentration of 50–100 nM) in living cells [82]. Substantial gain in sensitivity was further obtained by combining the use of a split Broccoli
aptamer and a catalytic hairpin assembly amplification circuit
(a molecular amplification circuit derived from RNA nanotechnology). The resulting CHARGE technology (Fig. 5f) allows detecting the presence of as few as 2.5 nM of target RNAs in live cells in a
digital manner (i.e., informs on the presence or absence of the
target) [83]. Last but not least, split aptamers can also be expressed
in cells and used to detect RNA-RNA interactions [84].
3.4 In Vitro Detection
of RNA
The key implication of miRs in various diseases and disorders has
stimulated the development of sensitive in vitro detection technologies for diagnostic applications. In this view, light-up RNA aptamers offer the great advantage of allowing to design cheap, labelfree, and sensitive assays. Following the same concepts as for livecell RNA imaging, the first generation of in vitro RNA sensors
exploited transiently destabilized aptamers (e.g., PANDAN technology [75]) and split aptamer [81] strategies. However, the
micromolar to high nanomolar sensitivity of these technologies
was insufficient to detect low-abundant RNAs suggesting that an
amplification step may be necessary. Several RNA-based molecular
amplification circuits, including CHARGE, have been developed
for in vitro applications [83, 85, 86] and could be used to increase
detection sensitivity. However, an elegant alternative consisting of
using the amplification capacity of the in vitro transcription reaction
has recently been proposed and revealed to be extremely efficient
for the sensitive detection of nucleic acids [87, 88] and other classes
Light-Up RNA Aptamers
91