- After finding an appropriate cell, fine-tune the focus to an
appropriate position for observation of the target RNA. - Capture images (Fig.4) and movies.
4 Notes
- The expression plasmids and their sequence information are
available from the authors’ lab (http://www.chem.s.u-tokyo.
ac.jp/~analyt/en/home_en.html). - The dissociation constant of PUM-HD mutants to the recog-
nized 8-base RNA is largely dependent on the three RNA bases
at the 5^0 terminus [8]. When the three bases are 5^0 -UGU-3^0 ,
the dissociation constant is reported to be nanomolar, whereas
the three-base sequence is not UGU, the dissociation constant
is approximately 100 nM. In case of mPUMt, the initial three-
base sequence is UUA. The dissociation constant was 100 nM. - A short incubation time after transfection of the TERRA probe
is recommended to avoid excess expression of the probe. Given
the irreversible reaction of EGFP reconstitution, overexpres-
sion of the probe molecule results in accumulation of recon-
stituted EGFP, which outnumbers TERRA and which
generates a severe background fluorescence signal in the obser-
vation. Therefore, the expression level of the probe is expected
to be sufficiently small by making the incubation time after the
transfection much shorter than described in the transfection
reagent manufacturer’s protocols. - In oblique illumination, making the light thin results in a high
signal-to-noise ratio because background fluorescence from
out-of-focus regions is eliminated, whereas the visible field
area becomes small [22]. In case of our TERRA observation,
the target RNA localizes in the nucleus. We made the
Fig. 4Images of TERRA using present methods: (A) TERRA labeled with the present probe, (B) iRFP-TRF1 that
represent telomeres, (C) hnRNPA1-SNAP-TMR, and (D) merge image
Spatiotemporal Imaging of Single Telomeric-Repeat Containing RNA 345