should be transcribed in fusion to a tRNA [66] or an F30 scaffold
[67] that assist in the proper folding and providing resistance to
RNase degradation. An elegant alternative called Tornado was
recently proposed in which the aptamer is flanked by a pair of
ribozymes that undergo self-cleavage just after RNA synthesis leaving 5
0 and 3
0 extremities that are joined together by a cellular ligase,
thus releasing a circular RNA (racRNA) endowed with an extremely
long half-life [68]. To visualize these stabilized light-up RNA aptamers, they are usually expressed from strong promoters (i.e., T7
RNA polymerase promoter in prokaryotes and U6 polymerase III
promoter in eukaryotes) to demonstrate both proper aptamer
functionality in the cellular environment and capacity of the fluorogen to cross the plasma membrane [15, 18, 21, 27–32, 35, 43,
58]. Moreover, proper expression and integrity of the aptamer can
also be assessed by extracting and analyzing total RNA by gel
electrophoresis. Staining the gel with a fluorogen (DFHBI or
TO1-Biotin) then allows to specifically detect RNAs labeled with
the light-up aptamer [67, 69]. Yet, to be significant, the fluorogen/
aptamer pair should also enable imaging less abundant but more
biologically relevant RNAs.
3.2 Live-Cell RNA
Imaging Using
Light-Up Aptamers
Inserted into
the Target RNA
An RNA of interest (ROI) can be imaged by expressing it in fusion
with the light-up aptamer (Fig. 5b). This strategy was initially
applied to abundant eukaryotic small noncoding RNAs transcribed
by RNA polymerase III (pol. III). Indeed, labeling 5S rRNA and
7SK RNA with Spinach2 allowed visualizing them at the expected
location (i.e., diffuse in the cytoplasm and in nuclear speckles,
respectively) [58]. Moreover, using a construct in which 60 CGG
repeats (the hallmark of some neurodegenerative diseases) were
fused to Spinach2 allowed to recapitulate the aggregation process
seen during the disease and to devise a drug screening strategy.
Other small pol. III transcripts (i.e., 5S rRNA, U6 RNA, a box
C/D scaRNA, or a tRNA) were also successfully detected at the
expected location upon labeling with Mango-II [43] or Corn
[21]. Extending this imaging strategy to less abundant pol. II
transcripts (mRNAs and microRNAs) turned to be more complex
because of the only moderate brightness and photostability of these
first set of probes (see above). Indeed, it was possible to detect and
track STL1 mRNA fused to a single copy of Spinach aptamer in
yeast using a sophisticated microscopy and image analysis pipeline
[63]. However, whereas this work demonstrated the great potential
of light-up RNA aptamers, the complexity of the approach prevented its wide use. Significant gain in signal was obtained by using
an array of up to 64 tandem repeats of Spinach introduced into the
3
0 untranslated region of an ROI without affecting its life cycle
[70]. Using brighter systems like TMR-DN/SRB2 allowed detecting CFP mRNA labeled with only 15 repeats of the aptamer in
mammalian and GFP mRNA labeled with only 6 repeats in bacteria
Light-Up RNA Aptamers
89
[67] that assist in the proper folding and providing resistance to
RNase degradation. An elegant alternative called Tornado was
recently proposed in which the aptamer is flanked by a pair of
ribozymes that undergo self-cleavage just after RNA synthesis leaving 5
0 and 3
0 extremities that are joined together by a cellular ligase,
thus releasing a circular RNA (racRNA) endowed with an extremely
long half-life [68]. To visualize these stabilized light-up RNA aptamers, they are usually expressed from strong promoters (i.e., T7
RNA polymerase promoter in prokaryotes and U6 polymerase III
promoter in eukaryotes) to demonstrate both proper aptamer
functionality in the cellular environment and capacity of the fluorogen to cross the plasma membrane [15, 18, 21, 27–32, 35, 43,
58]. Moreover, proper expression and integrity of the aptamer can
also be assessed by extracting and analyzing total RNA by gel
electrophoresis. Staining the gel with a fluorogen (DFHBI or
TO1-Biotin) then allows to specifically detect RNAs labeled with
the light-up aptamer [67, 69]. Yet, to be significant, the fluorogen/
aptamer pair should also enable imaging less abundant but more
biologically relevant RNAs.
3.2 Live-Cell RNA
Imaging Using
Light-Up Aptamers
Inserted into
the Target RNA
An RNA of interest (ROI) can be imaged by expressing it in fusion
with the light-up aptamer (Fig. 5b). This strategy was initially
applied to abundant eukaryotic small noncoding RNAs transcribed
by RNA polymerase III (pol. III). Indeed, labeling 5S rRNA and
7SK RNA with Spinach2 allowed visualizing them at the expected
location (i.e., diffuse in the cytoplasm and in nuclear speckles,
respectively) [58]. Moreover, using a construct in which 60 CGG
repeats (the hallmark of some neurodegenerative diseases) were
fused to Spinach2 allowed to recapitulate the aggregation process
seen during the disease and to devise a drug screening strategy.
Other small pol. III transcripts (i.e., 5S rRNA, U6 RNA, a box
C/D scaRNA, or a tRNA) were also successfully detected at the
expected location upon labeling with Mango-II [43] or Corn
[21]. Extending this imaging strategy to less abundant pol. II
transcripts (mRNAs and microRNAs) turned to be more complex
because of the only moderate brightness and photostability of these
first set of probes (see above). Indeed, it was possible to detect and
track STL1 mRNA fused to a single copy of Spinach aptamer in
yeast using a sophisticated microscopy and image analysis pipeline
[63]. However, whereas this work demonstrated the great potential
of light-up RNA aptamers, the complexity of the approach prevented its wide use. Significant gain in signal was obtained by using
an array of up to 64 tandem repeats of Spinach introduced into the
3
0 untranslated region of an ROI without affecting its life cycle
[70]. Using brighter systems like TMR-DN/SRB2 allowed detecting CFP mRNA labeled with only 15 repeats of the aptamer in
mammalian and GFP mRNA labeled with only 6 repeats in bacteria
Light-Up RNA Aptamers
89