- The fluorogenic dyes RG-DN, TMR-DN, SR-DN, and TR-
DN show 6-, 73-, 56-, and 15-fold fluorescence enhancement
upon binding to DNB, respectively, and the dissociation con-
stants are calculated to be 4.480.60μM, 0.350.05μM,
0.800.10μM, and 18.01.8μM, respectively. The best dye
for DNB aptamer is TMR-DN, because it shows the highest
fluorescence turn-on ratio and has the lowest KD. - To image RNA in bacterial cells, we used several different
plasmids in this protocol. The first plasmid (pET-tRNA)
expresses only the tRNA scaffold and was used as a negative
control to evaluate the background fluorescence due to non-
specific binding of the fluorogenic dyes to cellular biomole-
cules. pET-SRB2 and pET-DNB plasmids contain SRB-2 and
DNB aptamers, respectively, embedded in a tRNA scaffold
placed between T7 promoter and T7 terminator sequences
(seeFig. 4b).
Fig. 7Imaging of GFP mRNA in live bacteria. Bacteria were transformed with pET-GFP, pET-GFP-4xDNB, or
pET-GFP-8xDNB and transcription was induced with IPTG. Bacteria were incubated with 1μM of TMR-DN (red)
for 10 min and both GFP protein (green) and GFP mRNA (red) were imaged at 37C. GFP mRNA was found to
localize at the poles of the bacteria. Scale bar, 3μm
300 Murat Sunbul et al.