acids participating in RNA recognition [8](seeFig. 1d and
Note2).- In addition to the usage of PUM-HD, employment of a fluo-
rescent protein reconstitution technique is important for the
design of PUM-HD based probes for sensitive and precise
monitoring of the target RNA. Fluorescence from excess
probes that is not binding to the target RNA causes a high
background signal and disturb detection of the target RNA.
The fluorescent protein reconstitution technique is a strategy
to reduce the background fluorescence signal from excess
probe molecules [18, 19]. - For the fluorescent protein reconstitution technique, N-
terminal and C-terminal fragments of EGFP (GN and GC,
respectively) are used. When the two fragments become mutu-
ally close, they cause a reconstitution reaction to form the
structure of the full-length protein and regain their original
fluorescence [19–21]. Using this technique, RNA probes can
be designed to emit fluorescence just upon binding to the
target RNA. Although the approach on fluorescent protein
reconstitution is effective to eliminate background fluorescence
and to improve the signal-to-background ratio in the obtained
images, this approach has some limitations. An important limi-
tation is the slow rate of the reconstitution reaction. The
reaction of the pair of EGFP fragments, from mutual approach
Fig. 1Crystal structure and RNA recognition of PUM-HD. (A) Crystal structure of PUM-HD. (B) Enlarged images
of the amino acids–RNA interacting portions. (C) Combination of RNA recognizing amino acids in the eight
repeated motifs in the wild-type PUM-HD. (D) The universal code of RNA recognizing amino acids at the third,
fourth, and seventh positions in the second helix, and recognized the RNA bases. X indicates positions at
which any amino acid is acceptable
Spatiotemporal Imaging of Single Telomeric-Repeat Containing RNA 341