[29]. The number of aptamer repeats was even further recently
decreased by labeling β-actin mRNA with only four copies of the
Riboglow aptamer while still being able to observe RNA relocation
in stress granules with a signal quality outperforming the MS2-GFP
method [32]. Recently, direct labeling of GFP mRNA by only five
copies of the very bright SiRA aptamer allowed to detect RNA
synthesis in bacteria and to gain in imaging accuracy using STED
super-resolution imaging [30].
Recently, it has been reported that the direct insertion of the
probe may lead to folding interference between the labeled mRNA
and the light-up aptamer, as in the case of eGFP mRNA labeled
with SRB2 aptamer [71]. Yet, such an adverse effect can be avoided
by inserting a Dicer cleavage site between the mRNA and the
aptamer so that, upon synthesis, Dicer physically separates both
molecules and thereby restores the aptamer folding capacity. However, while still informative on the synthesis of the ROI, this
approach compromises the chances to track the ROI in the cell.
Another way of avoiding folding interference while reducing the
required genetics is to use probes acting in trans.
3.3 Live-Cell RNA
Imaging Using
Light-Up Aptamers
Acting In Trans
Besides the direct insertion of a light-up aptamer into the sequence
of an ROI, the labeling can also be performed in trans (Fig. 5c–f).
A first engineering strategy consists of transiently destabilizing a key
structural element (e.g., a stem close to the fluorogen-binding site)
of the aptamer by shortening and/or mutating it while appending
sequences complementary to the ROI near the destabilized stem
(Fig. 5c). In its free state, the RNA probe is unable to interact with
the fluorogen and cannot form a fluorescent complex. However,
upon interaction with the ROI, the light-up domain of the probe
recovers its “active” folding together with its capacity to bind its
fluorogen, resulting in the fluorescent labeling of the ROI. Such
approach was successfully applied to endogenous mRNA visualization using engineered versions of the BHQ-1-binding aptamer
[72] and of Spinach [73]. Recently, improved designs of Broccoliderived probes with optimized sequences have been proposed and
were shown to display superior performances to detect mRNA
in vitro [61, 74]. Yet, these probes still need to be validated in
live cells. Similar design was also exploited for the development of a
probe called PANDAN aimed at detecting microRNA (miR),
though this probe was not evaluated in living cells either [75].
Besides stem stabilization, aptamer activation can also take
place through more important structural remodeling involving
strand displacements (Fig. 5d). For instance, in FASTmiR technology, an engineered version of Spinach, is trapped in an inactive form
and the successful binding of a target miR to a sensing domain
induces, both in vitro and in live cells, several strand displacements
eventually leading to the release of an active Spinach domain
[76]. Substantial gain in sensitivity was later achieved by
90
Michael Ryckelynck
decreased by labeling β-actin mRNA with only four copies of the
Riboglow aptamer while still being able to observe RNA relocation
in stress granules with a signal quality outperforming the MS2-GFP
method [32]. Recently, direct labeling of GFP mRNA by only five
copies of the very bright SiRA aptamer allowed to detect RNA
synthesis in bacteria and to gain in imaging accuracy using STED
super-resolution imaging [30].
Recently, it has been reported that the direct insertion of the
probe may lead to folding interference between the labeled mRNA
and the light-up aptamer, as in the case of eGFP mRNA labeled
with SRB2 aptamer [71]. Yet, such an adverse effect can be avoided
by inserting a Dicer cleavage site between the mRNA and the
aptamer so that, upon synthesis, Dicer physically separates both
molecules and thereby restores the aptamer folding capacity. However, while still informative on the synthesis of the ROI, this
approach compromises the chances to track the ROI in the cell.
Another way of avoiding folding interference while reducing the
required genetics is to use probes acting in trans.
3.3 Live-Cell RNA
Imaging Using
Light-Up Aptamers
Acting In Trans
Besides the direct insertion of a light-up aptamer into the sequence
of an ROI, the labeling can also be performed in trans (Fig. 5c–f).
A first engineering strategy consists of transiently destabilizing a key
structural element (e.g., a stem close to the fluorogen-binding site)
of the aptamer by shortening and/or mutating it while appending
sequences complementary to the ROI near the destabilized stem
(Fig. 5c). In its free state, the RNA probe is unable to interact with
the fluorogen and cannot form a fluorescent complex. However,
upon interaction with the ROI, the light-up domain of the probe
recovers its “active” folding together with its capacity to bind its
fluorogen, resulting in the fluorescent labeling of the ROI. Such
approach was successfully applied to endogenous mRNA visualization using engineered versions of the BHQ-1-binding aptamer
[72] and of Spinach [73]. Recently, improved designs of Broccoliderived probes with optimized sequences have been proposed and
were shown to display superior performances to detect mRNA
in vitro [61, 74]. Yet, these probes still need to be validated in
live cells. Similar design was also exploited for the development of a
probe called PANDAN aimed at detecting microRNA (miR),
though this probe was not evaluated in living cells either [75].
Besides stem stabilization, aptamer activation can also take
place through more important structural remodeling involving
strand displacements (Fig. 5d). For instance, in FASTmiR technology, an engineered version of Spinach, is trapped in an inactive form
and the successful binding of a target miR to a sensing domain
induces, both in vitro and in live cells, several strand displacements
eventually leading to the release of an active Spinach domain
[76]. Substantial gain in sensitivity was later achieved by
90
Michael Ryckelynck