RNA Detection

7. For background correction, Fiji/ImageJ image analysis soft-
   ware is used to manually pick a surface area where noE. coli
   cells are attached (seeNote 15) and the mean fluorescent signal
   obtained is subtracted from the whole image.

3.4 Dual-Color RNA
Imaging

There is an increasing demand to develop methods that allow for

imaging of multiple RNAs simultaneously in living cells, which

facilitates studies on RNA co-localizations and RNA-RNA interac-

tions. To image two RNAs simultaneously inside live bacterial cells,

the quencher-binding aptamer (DNB) was combined with a

fluorophore-binding aptamer (SRB-2). Using two cognate pairs

of fluorogenic dye–aptamer, namely, SR-MN–SRB-2 and RG-

DN–DNB, we were able to successfully image two ROIs in liveE.

coli(seeNote 16).

1. The expression plasmid (pET-SRB2-DNB) and control plas-
   mids (pET-DNB and pET-SRB2) are transformed into BL21
   Star™(DE3) competent E. coli cells and transcription is
   induced as mentioned previously in Subheading3.2.


2. The glass slides are coated with poly-D-lysine as described in
   Subheading3.3.1.
   3.E. colicells expressing the SRB-2 and DNB aptamers are
   prepared for imaging as described in Subheading3.3.2except
   for the following steps.


3. After washing the unattachedE. colicells (step 6of Subhead-
   ing3.3.2), the wells are incubated with 300μL of imaging
   solution containing 1μM of SR-MN and 1μM of RG-DN.


4. Aptamers in live bacteria are imaged as mentioned in Subhead-
   ing3.3.3. Filter sets for SR-MN dye are as same as the ones for
   SR-DN (seeFig. 6).

3.5 mRNA Imaging
with Tandem Repeats
of DNB

Imaging mRNA in live bacteria can be quite challenging since they

are not necessarily very abundant and their half-life can be very

short, on the order of several minutes. To this end, tandem repeats

of DNB aptamer can be very advantageous to increase the signal-

to-noise ratio in imaging. Here, we use two different plasmids to

label GFP mRNA; one has only four repeats of DNB (pET-GFP-

4xDNB), while the other one contains 8 repeats of DNB (pET-

GFP-8xDNB).

1. Both plasmids and pET-GFP (control plasmid) without DNB
   aptamer are transformed into BL21 Star™(DE3) competent
   E. colicells and transcription is induced as mentioned previ-
   ously in Subheading3.2.


2. The glass slides are coated with poly-D-lysine as described in
   Subheading3.3.1.

298 Murat Sunbul et al.