RNA Detection

7. Incubator and shaker at 37C.


8. Spectrophotometer.

2.4 Microscopy and
Image Analysis

1. 8-well Nunc™Lab-Tek™II chambered cover glass (Thermo
   Scientific).


2. 50μg/mL poly-D-lysine: poly-D-lysine hydrobromide (molec-
   ular weight 30,000–70,000 g/mol, lyophilized powder) is
   dissolved in PBS at a concentration of 1 mg/mL and kept as
   a stock solution at 4C. 50μg/mL poly-D-lysine is prepared
   freshly by diluting the stock with dH 2 O(seeNote 8).


3. Cells transformed with either a plasmid carrying the RNA of
   interest fused to the aptamer tag or a plasmid carrying the RNA
   of interest without the aptamer tag (negative control).


4. 1 M isopropyl-β-D-thio-galactopyranoside (IPTG).


5. Live-cell imaging solution (Invitrogen): 20 mM HEPES,
   140 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl 2 , and 1 mM
   MgCl 2 , pH 7.4. This solution is always supplemented with
   additional 5 mM MgCl 2 and 20 mM glucose and will be
   referred as “imaging solution” in the following sections.


6. Fluorogenic dyes: 100μM of RG-DN, TMR-DN, SR-DN,
   TR-DN, and SR-MN (all of them dissolved in DMSO).


7. An automated wide field epifluorescence microscope, e.g., a
   Nikon TiE equipped with a Nikon 100Plan Apo lambda oil
   immersion objective (NA 1.45), a cooled CCD Hamamatsu
   Orca-AG camera and a TokaiHit INU ZILCS incubator box.


8. Incubator and a shaker at 37C.


9. Fiji image analysis software (https://fiji.sc/).

3 Methods

3.1 Expression
Plasmids

1. Both SRB-2 (54-nucleotide) and DNB (75-nucleotide) apta-
   mers are quite small and do not require special scaffolds for
   RNA imaging in bacterial cells. Therefore, they can be directly
   fused to either 3^0 or 5^0 of the RNA of interest during PCR by
   using forward and reverse primers one of which carries the
   aptamer sequence (seeNote 6).


2. Restriction sites should also be included in the primer sequence
   and the PCR product can be cloned into the multiple cloning
   site (MCS) of pET28 plasmid (seeNote 6).


3. (Optional) To image less stable and low abundant RNAs tan-
   dem repeats of SRB-2 or DNB aptamers can be utilized which
   increases the signal-to-noise ratio (seeNote 5).


4. UsingappropriaterestrictionenzymessubstitutetheGFPcoding
   sequence (cds) with the cds of the RNA of interest in pET-GFP-
   4xDNB or pET-GFP-8xDNB plasmids (seeFig. 4d,Note 5).

294 Murat Sunbul et al.