Interestingly, Mango-III displays important differences with the
other Mango aptamers both at the sequence and the structure
level (see below). Moreover, Mango-III dominated the pool at
the end of the screening process, whereas it was completely missed
during the previous hand screenings [14]. Taken together, the case
of Mango-III demonstrates how powerful the combined use of
SELEX and μIVC can be at identifying efficient light-up RNA
aptamers.
Finally, also microarrays were used for in vitro functional
screening of aptamers [44–46]. Upon DNA spotting onto a surface, genes are transcribed and captured either onto a functionalized coverslip [46] or into a dedicated micro-chamber [44]. Then,
the RNA is incubated with the fluorogen and the array imaged.
Moreover, using a microfluidic device it became possible to keep
the array under perfusion of liquid and to vary the concentration of
the fluorogen (or other compounds) while collecting thermodynamic data on the system [44]. Whereas array-based technologies
operate at much lower throughput than μIVC and are therefore less
efficient at isolating optimal light-up aptamers from large pools, the
possibility to directly associate each phenotype to the encoding
genotype makes these techniques particularly well suited for the
fine functional characterization of light-up aptamers via the screening of comprehensive single (if not double)-point mutant libraries.
2.2.2 Structure-Assisted
Characterization
and Optimization
of Light-Up Aptamers
Once a light-up aptamer has been identified, the most efficient way
to understand the molecular mechanism driving the recognition
and the activation of the fluorogen is to solve the crystal structure
of the fluorogen/aptamer complex. Moreover, such structural
characterization is highly valuable to further engineer the system
(e.g., to develop biosensors and supramolecular assemblies, see
below). Thus, nearly half of the systems presented in Table 1 have
been crystalized and their structure has been solved. Even though
each aptamer adopts an idiosyncratic folding, they all share the
presence of an extended platform made of a base-triple [47], a
base-quadruple [48], or even a G-quartet [49–54] that accommodates the fluorogen [53]. Fluorogens are polycyclic compounds
and their optimal activation is usually obtained by holding them
in planar conformation while properly confining them within a
binding pocket.
On a structural point of view, the original Mango aptamer
possesses one of the simplest fluorogen-binding pockets, which
consists of a G-quartet surmounted by three unpaired nucleotides
(two As and a U) that belong to G-quadruplex propellers (Fig. 4a)
[52]. As a consequence, one face of the fluorogen is still largely
exposed to the solvent, explaining in part the low QY of the
complex (Table 1). Moreover, both cycles of the TO1 fluorogen
are rotated relative to each other instead of being coplanar as
Light-Up RNA Aptamers
83
other Mango aptamers both at the sequence and the structure
level (see below). Moreover, Mango-III dominated the pool at
the end of the screening process, whereas it was completely missed
during the previous hand screenings [14]. Taken together, the case
of Mango-III demonstrates how powerful the combined use of
SELEX and μIVC can be at identifying efficient light-up RNA
aptamers.
Finally, also microarrays were used for in vitro functional
screening of aptamers [44–46]. Upon DNA spotting onto a surface, genes are transcribed and captured either onto a functionalized coverslip [46] or into a dedicated micro-chamber [44]. Then,
the RNA is incubated with the fluorogen and the array imaged.
Moreover, using a microfluidic device it became possible to keep
the array under perfusion of liquid and to vary the concentration of
the fluorogen (or other compounds) while collecting thermodynamic data on the system [44]. Whereas array-based technologies
operate at much lower throughput than μIVC and are therefore less
efficient at isolating optimal light-up aptamers from large pools, the
possibility to directly associate each phenotype to the encoding
genotype makes these techniques particularly well suited for the
fine functional characterization of light-up aptamers via the screening of comprehensive single (if not double)-point mutant libraries.
2.2.2 Structure-Assisted
Characterization
and Optimization
of Light-Up Aptamers
Once a light-up aptamer has been identified, the most efficient way
to understand the molecular mechanism driving the recognition
and the activation of the fluorogen is to solve the crystal structure
of the fluorogen/aptamer complex. Moreover, such structural
characterization is highly valuable to further engineer the system
(e.g., to develop biosensors and supramolecular assemblies, see
below). Thus, nearly half of the systems presented in Table 1 have
been crystalized and their structure has been solved. Even though
each aptamer adopts an idiosyncratic folding, they all share the
presence of an extended platform made of a base-triple [47], a
base-quadruple [48], or even a G-quartet [49–54] that accommodates the fluorogen [53]. Fluorogens are polycyclic compounds
and their optimal activation is usually obtained by holding them
in planar conformation while properly confining them within a
binding pocket.
On a structural point of view, the original Mango aptamer
possesses one of the simplest fluorogen-binding pockets, which
consists of a G-quartet surmounted by three unpaired nucleotides
(two As and a U) that belong to G-quadruplex propellers (Fig. 4a)
[52]. As a consequence, one face of the fluorogen is still largely
exposed to the solvent, explaining in part the low QY of the
complex (Table 1). Moreover, both cycles of the TO1 fluorogen
are rotated relative to each other instead of being coplanar as
Light-Up RNA Aptamers
83