of targets (see below). Briefly, the target miR interacts with two
DNA molecules (one containing the T7 RNA polymerase promoter and the second containing the sequence coding for Spinach
aptamer) and drives their specific ligation to form a full-length
transcription template. Then, each template is transcribed into a
large number of Spinach aptamers that become fluorescent in the
presence of DFHBI(-1T). Such amplification-based detections
allow the specific detection of miR with a low femtomolar limit of
detection. In the latest format of these technologies, this limit was
pushed down to the attomolar range (5 aMol) by replacing the
ligation step by a primer extension coupled with a strand displacement amplification (SDA), adding a second amplification step to
the process [89].
Being able to detect RNA in vitro allows also to set up new
screening and analytical pipelines in which the in vitro transcription
process can be monitored either alone [90] or in tandem with
in vitro translation [91–93]. This ability to independently monitor
RNA and protein (e.g., using fluorescent protein) synthesis permits
to finely tune gene expression rates for applications in synthetic
biology, for instance. Light-up RNA aptamers can also be used as
reporters to aid in the development of catalytic RNAs endowed
with self-cleaving [94] or RNA-modifying activities [95].
Finally, light-up RNA aptamers also offer the great opportunity
to assist and validate the design of supramolecular assemblies in
RNA nanotechnology. RNA nanotechnology aims at designing
programmable molecular circuits (such as the catalytic hairpin
assemblies introduced above) and supramolecular assemblies to
achieve complex functions (e.g., computation using logic gates,
channeling of catalysts on functionalized surfaces, drug delivery)
in vitro or in living cells by genetically encoding the system. For
instance, light-up aptamers can be used as output signal of a molecular circuit aiming at amplifying a signal [85, 96] or analyzing
several inputs using logic gates as complex as half-adders
[97]. Incorporating a monolithic light-up aptamer into supramolecular assemblies is also frequently used to assess that RNA elements grafted onto the assembly preserve their function as well as
to track these nano-objects [98, 99]. Last but not least, split versions of MGA [100–103], Spinach [102, 104], or Broccoli
[105, 106] revealed to be extremely useful to validate the proper
assembly of multistrand RNA nanoparticles forming a variety of
tiles [102, 103, 105] but also more elaborated shapes like cubes
[100] and rings [104], just to name a few examples.
3.5 Detection
of Other Biological
Molecules and Ions
Beyond RNA detection, light-up RNA aptamers also found a wide
range of applications allowing to specifically detect and quantify
several other types of target. For instance, by inserting Spinach2
into sgRNAs, it is possible to precisely localize specific loci on
genomic DNA using CRISPR-display technology [107]. In a
92
Michael Ryckelynck
DNA molecules (one containing the T7 RNA polymerase promoter and the second containing the sequence coding for Spinach
aptamer) and drives their specific ligation to form a full-length
transcription template. Then, each template is transcribed into a
large number of Spinach aptamers that become fluorescent in the
presence of DFHBI(-1T). Such amplification-based detections
allow the specific detection of miR with a low femtomolar limit of
detection. In the latest format of these technologies, this limit was
pushed down to the attomolar range (5 aMol) by replacing the
ligation step by a primer extension coupled with a strand displacement amplification (SDA), adding a second amplification step to
the process [89].
Being able to detect RNA in vitro allows also to set up new
screening and analytical pipelines in which the in vitro transcription
process can be monitored either alone [90] or in tandem with
in vitro translation [91–93]. This ability to independently monitor
RNA and protein (e.g., using fluorescent protein) synthesis permits
to finely tune gene expression rates for applications in synthetic
biology, for instance. Light-up RNA aptamers can also be used as
reporters to aid in the development of catalytic RNAs endowed
with self-cleaving [94] or RNA-modifying activities [95].
Finally, light-up RNA aptamers also offer the great opportunity
to assist and validate the design of supramolecular assemblies in
RNA nanotechnology. RNA nanotechnology aims at designing
programmable molecular circuits (such as the catalytic hairpin
assemblies introduced above) and supramolecular assemblies to
achieve complex functions (e.g., computation using logic gates,
channeling of catalysts on functionalized surfaces, drug delivery)
in vitro or in living cells by genetically encoding the system. For
instance, light-up aptamers can be used as output signal of a molecular circuit aiming at amplifying a signal [85, 96] or analyzing
several inputs using logic gates as complex as half-adders
[97]. Incorporating a monolithic light-up aptamer into supramolecular assemblies is also frequently used to assess that RNA elements grafted onto the assembly preserve their function as well as
to track these nano-objects [98, 99]. Last but not least, split versions of MGA [100–103], Spinach [102, 104], or Broccoli
[105, 106] revealed to be extremely useful to validate the proper
assembly of multistrand RNA nanoparticles forming a variety of
tiles [102, 103, 105] but also more elaborated shapes like cubes
[100] and rings [104], just to name a few examples.
3.5 Detection
of Other Biological
Molecules and Ions
Beyond RNA detection, light-up RNA aptamers also found a wide
range of applications allowing to specifically detect and quantify
several other types of target. For instance, by inserting Spinach2
into sgRNAs, it is possible to precisely localize specific loci on
genomic DNA using CRISPR-display technology [107]. In a
92
Michael Ryckelynck