photostability of the FP-mimicking fluorogens was recently
reached with the isolation of Corn, a DFHO-binding aptamer
protecting its fluorogen from the rapid photoisomerization
encountered with Spinach and Broccoli families [21]. Interestingly,
this impressive gain in photostability was not attributable to the
fluorogen itself (which does not display such a stability when bound
to Broccoli aptamers) but rather to the way the aptamer accommodates it. Indeed, the crystal structure of DFHO/Corn revealed
that the RNA is organized around a G-quadruplex and that the
DFHO is actually accommodated between the apical quartet of two
monomers forming a complex with a fluorogen:aptamer stoichiometry of 1:2. (Fig. 4g) [62]. As a consequence, the DFHO is
maintained in an emissive planar conformation by being sandwiched between both G-quartets while five unpaired adenines further restrict possible movement. Altogether, these elements
prevent photoisomerization to take place and confer to DFHO/
Corn a very high photostability.
3 Imaging and Sensing Applications Using Light-Up RNA Aptamers
As highlighted in the introduction of this chapter, one of the main
motivations in developing fluorogens and light-up RNA aptamers
was the possibility foreseen to directly monitor RNA synthesis, as
well as the movement and location of RNA molecules within living
cells, while overcoming limitations of RBP-FPs. The strong toxicity
of malachite green for yeast and mammalian cells [38] largely
prevented its use as a fluorogen for live-cell RNA imaging. On the
contrary, all the other above-introduced fluorogens were found to
be nontoxic, deprived of nonspecific interaction with cell components and most of them were further found to be cell membrane
permeable. Therefore, most of these systems are well suited for livecell imaging applications. Moreover, since nucleic acids are molecules highly amenable to engineering, these aptamers were rapidly
converted into a variety of sensors with a wide application spectrum
both in live cells and in vitro.
3.1 General
Considerations Before
Starting Live-Cell
Applications
So far, DFHBI(-1T) has been the most broadly used fluorogen, as
it is commercially available from several companies and is able to
enter various cell types such as bacteria [18], yeast [63, 64], algae
[65], and mammalian cells [18, 35, 58]. However, other fluorogens like DFHO and TO1-Biotin are also known to be cell permeable and are now commercialized (by Lucerna and Applied
Biological Material, respectively), which will further ease their
wide use in the near future. Because of the short half-life of short
RNAs in cells and because of limited folding capacity, fluorescence
can hardly be observed upon expressing Spinach or Broccoli family
aptamers as free RNA molecules (Fig. 5a). Instead, aptamer RNAs
Light-Up RNA Aptamers
87
reached with the isolation of Corn, a DFHO-binding aptamer
protecting its fluorogen from the rapid photoisomerization
encountered with Spinach and Broccoli families [21]. Interestingly,
this impressive gain in photostability was not attributable to the
fluorogen itself (which does not display such a stability when bound
to Broccoli aptamers) but rather to the way the aptamer accommodates it. Indeed, the crystal structure of DFHO/Corn revealed
that the RNA is organized around a G-quadruplex and that the
DFHO is actually accommodated between the apical quartet of two
monomers forming a complex with a fluorogen:aptamer stoichiometry of 1:2. (Fig. 4g) [62]. As a consequence, the DFHO is
maintained in an emissive planar conformation by being sandwiched between both G-quartets while five unpaired adenines further restrict possible movement. Altogether, these elements
prevent photoisomerization to take place and confer to DFHO/
Corn a very high photostability.
3 Imaging and Sensing Applications Using Light-Up RNA Aptamers
As highlighted in the introduction of this chapter, one of the main
motivations in developing fluorogens and light-up RNA aptamers
was the possibility foreseen to directly monitor RNA synthesis, as
well as the movement and location of RNA molecules within living
cells, while overcoming limitations of RBP-FPs. The strong toxicity
of malachite green for yeast and mammalian cells [38] largely
prevented its use as a fluorogen for live-cell RNA imaging. On the
contrary, all the other above-introduced fluorogens were found to
be nontoxic, deprived of nonspecific interaction with cell components and most of them were further found to be cell membrane
permeable. Therefore, most of these systems are well suited for livecell imaging applications. Moreover, since nucleic acids are molecules highly amenable to engineering, these aptamers were rapidly
converted into a variety of sensors with a wide application spectrum
both in live cells and in vitro.
3.1 General
Considerations Before
Starting Live-Cell
Applications
So far, DFHBI(-1T) has been the most broadly used fluorogen, as
it is commercially available from several companies and is able to
enter various cell types such as bacteria [18], yeast [63, 64], algae
[65], and mammalian cells [18, 35, 58]. However, other fluorogens like DFHO and TO1-Biotin are also known to be cell permeable and are now commercialized (by Lucerna and Applied
Biological Material, respectively), which will further ease their
wide use in the near future. Because of the short half-life of short
RNAs in cells and because of limited folding capacity, fluorescence
can hardly be observed upon expressing Spinach or Broccoli family
aptamers as free RNA molecules (Fig. 5a). Instead, aptamer RNAs
Light-Up RNA Aptamers
87