different way, fusing Spinach to the PP7 RNA sequence enables to
bring the aptamer in close proximity to a PP7-mCherry fusion
protein and, this way, to characterize RNA/protein interaction
(e.g., affinity measurement) by monitoring FRET (Fo ¨rster resonance emission transfer) between DFHBI/Spinach and mCherry
fluorophores [108]. Specific protein/RNA interactions can also be
established by fusing the light-up RNA to another aptamer that
specifically targets a protein. This concept was applied to monitor
EGFR (epidermal growth factor receptor) internalization by labeling the receptor with an EGFR-binding aptamer fused to the
DIR2s aptamer [17]. As in the case described above for RNA
detection, proteins can also be detected by using a bipartite probe
made of a transiently destabilized light-up aptamer domain fused to
a second aptamer binding specifically to the protein of interest
(Fig. 5g). As before, the specific interaction between the protein
and the sensing aptamer stabilizes the overall structure of the probe
and restores the fluorogen-binding capacity of the light-up aptamer
moiety. Such allosteric probes were successfully derived from Spinach and applied to the detection of a variety of proteins synthesized
in E. coli [109]. Whereas such a direct labeling is particularly well
suited to monitor protein synthesis in living cells, it does not offer
sufficient sensitivity to detect low-abundant protein targets as
potentially required for diagnostic purposes. Yet, combining conventional immunoassays like the proximity ligation assay (PLA) and
ELISA with the Spinach transcription-based amplification strategy
introduced above allowed to devise ultrasensitive assays able to
detect proteins present at picomolar [110] and attomolar [111]
concentrations. Interestingly, applying the same concept to the
detection of whole bacteria allowed to devise an assay able to
specifically identify the presence of as few as 77 Staphylococcus
aureus per mL of food samples [112]. Light-up aptamers can also
be used to monitor the activity of enzymes, such as Dicer [113],
telomerase [114], or RNA-modifying enzymes [115]. These
fluorogenic assays are compatible with high-throughput screening
and are therefore suited for drug discovery applications, for
example.
Light-up RNA aptamers also served as building blocks in the
design of metabolite biosensors permitting to monitor the accumulation of a target metabolite in real time and in a noninvasive
manner. Such genetically encoded biosensors were developed, for
instance, to detect SAM [116], FMN [117], TPP, cyclic AMP
[118], as well as various types of cyclic dinucleotides [119–
125]. In general, these sensors are based on the transient destabilization strategy (Fig. 5h) similar to that described above for RNA
and protein detection. These sensors are usually developed by using
a trial-and-error approach, but optimal biosensors can also be
Light-Up RNA Aptamers
93
bring the aptamer in close proximity to a PP7-mCherry fusion
protein and, this way, to characterize RNA/protein interaction
(e.g., affinity measurement) by monitoring FRET (Fo ¨rster resonance emission transfer) between DFHBI/Spinach and mCherry
fluorophores [108]. Specific protein/RNA interactions can also be
established by fusing the light-up RNA to another aptamer that
specifically targets a protein. This concept was applied to monitor
EGFR (epidermal growth factor receptor) internalization by labeling the receptor with an EGFR-binding aptamer fused to the
DIR2s aptamer [17]. As in the case described above for RNA
detection, proteins can also be detected by using a bipartite probe
made of a transiently destabilized light-up aptamer domain fused to
a second aptamer binding specifically to the protein of interest
(Fig. 5g). As before, the specific interaction between the protein
and the sensing aptamer stabilizes the overall structure of the probe
and restores the fluorogen-binding capacity of the light-up aptamer
moiety. Such allosteric probes were successfully derived from Spinach and applied to the detection of a variety of proteins synthesized
in E. coli [109]. Whereas such a direct labeling is particularly well
suited to monitor protein synthesis in living cells, it does not offer
sufficient sensitivity to detect low-abundant protein targets as
potentially required for diagnostic purposes. Yet, combining conventional immunoassays like the proximity ligation assay (PLA) and
ELISA with the Spinach transcription-based amplification strategy
introduced above allowed to devise ultrasensitive assays able to
detect proteins present at picomolar [110] and attomolar [111]
concentrations. Interestingly, applying the same concept to the
detection of whole bacteria allowed to devise an assay able to
specifically identify the presence of as few as 77 Staphylococcus
aureus per mL of food samples [112]. Light-up aptamers can also
be used to monitor the activity of enzymes, such as Dicer [113],
telomerase [114], or RNA-modifying enzymes [115]. These
fluorogenic assays are compatible with high-throughput screening
and are therefore suited for drug discovery applications, for
example.
Light-up RNA aptamers also served as building blocks in the
design of metabolite biosensors permitting to monitor the accumulation of a target metabolite in real time and in a noninvasive
manner. Such genetically encoded biosensors were developed, for
instance, to detect SAM [116], FMN [117], TPP, cyclic AMP
[118], as well as various types of cyclic dinucleotides [119–
125]. In general, these sensors are based on the transient destabilization strategy (Fig. 5h) similar to that described above for RNA
and protein detection. These sensors are usually developed by using
a trial-and-error approach, but optimal biosensors can also be
Light-Up RNA Aptamers
93