- For dual-color RNA imaging experiments, we created a single
plasmid (pET-SRB2-DNB) which expresses both SRB-2 and
DNB aptamers embedded in the tRNA scaffold from their own
independent T7 promoter/terminator systems (seeFig. 4c). It
is also possible to use two different plasmids for the same
purpose: one transcribes an ROI-1-SRB2 fusion and the
other one transcribes an ROI-2-DNB fusion. They should be
compatible to each other and carry different antibiotic selec-
tion markers. Bacteria can be transformed with both of the
plasmids and expression of both transcripts can be induced by
the addition of IPTG. - We prepared two plasmids (pET-GFP-4xDNB or pET-GFP-
8xDNB) for the imaging of GFP mRNA in live bacteria. In
these plasmids, GFP is expressed from the T7/lacO promoter
and tandem repeats (4 or 8 times) of DNB were fused to the 3^0
UTR region of the GFP gene right after the stop codon (see
Fig.4d). Any RNA of interest can be substituted for GFP by
taking advantage of the restriction enzyme sites before and
after the GFP sequence. Alternatively, tandem repeats of
DNB can be cut out from the plasmids (pET-GFP-4xDNB or
pET-GFP-8xDNB) by using appropriate restriction enzymes,
purified on an agarose gel, and used as a cassette, which can be
cloned into any plasmid carrying the gene of interest. - Since SRB-2 and DNB aptamers are quite small in size, they can
easily be fused to the ROI during PCR by using a relatively
long primer. Primer design for the PCR depends if the aptamer
will be fused to the 3^0 or 5^0 end of the ROI. For example, if the
aptamer is to be fused to the 5^0 end of the ROI, forward primer
should contain an appropriate restriction site sequence, either
SRB-2 or DNB aptamer sequence, and a gene specific
sequence, respectively (5^0 –3^0 direction). Whereas, the reverse
primer should contain an appropriate restriction site sequence
and a reverse complementary of the gene specific sequence,
respectively (5^0 –3^0 direction). If the aptamer is to be fused to
the 3^0 end of the ROI, forward primer should contain an
appropriate restriction site sequence and a gene specific
sequence, respectively (5^0 –3^0 direction), whereas the reverse
primer should contain an appropriate restriction site, the
reverse complementary of either SRB-2 or DNB aptamer, and
the reverse complementary of a gene specific sequence, respec-
tively (5^0 –3^0 direction). Finally, PCR product can be cloned into
any vector of interest (e.g., pET vectors) by using restriction
site digestion and ligation reactions. - The synthesized dyes for in vivo imaging applications have to
be extremely pure. Since the contact-quenched dyes are non-
fluorescent, any fluorescent impurity would dramatically
decrease the turn-on ratios.
Visualizing RNA with Fluorogenic Aptamers in vivo 301