- PCR purification kit (e.g., Qiagen).
- Restriction enzymes.
- DNA ligase.
- 1% agarose and gel electrophoresis equipment.
- 10 mg/mL ethidium bromide.
- 1TAE (Tris–acetate–EDTA) Buffer: 40 mM Tris–acetate and
1 mM EDTA at pH 8.3. - Mini-prep kit (Qiagen).
2.2
Fluorophore–Quencher
Conjugates
Fluorogenic dyes used in this study are not commercially available.
Therefore, they have to be synthesized and purified as described in
the literature [17, 23](seeNote 7).- 100μM of RG-DN (in DMSO), 100μM of TMR-DN (in
DMSO), 100μM of SR-DN (in DMSO), 100 ofμM SR-MN
(in DMSO), 100μM of TR-DN (in DMSO).
2.3 Bacterial Growth 1. BL21 Star™(DE3) (Invitrogen), and DH5α™(Invitrogen)
competentE. colistrains.
- 1 M isopropyl-β-D-thio-galactopyranoside (IPTG).
- 30 mg/mL kanamycin (Kan) in water.
- Autoclaved Luria–Bertani (LB) media: 10 g tryptone/L, 5 g
yeast extract/L, and 5 g NaCl/L. - LB-agar plates containing 30μg/mL of kanamycin.
- 50 mL baffled Erlenmeyer flask.
Fig. 4(a) A general scheme for a cloning vector for RNA imaging with aptamers. P denotes a promoter
sequence, ROI denotes an RNA of interest and T denotes a terminator sequence. A single copy or tandem
repeats of the aptamer should be fused to the ROI. (b) Cloning vector used for expressing tRNA (negative
control, pET-tRNA) or aptamers (SBR-2 or DNB) embedded in a tRNA scaffold (pET-SRB2 and pET-DNB). (c)
Cloning vector used for expressing both SRB-2 and DNB aptamers inE. coli(pET-SRB2-DNB). (d) A general
scheme of a cloning vector for mRNA imaging in bacteria (pET-GFP-4xDNB and pET-GFP-8xDNB)
Visualizing RNA with Fluorogenic Aptamers in vivo 293