Investigating the Effect of RNA Scaffolds on the Multicolor Fluorogenic Aptamer Pepper in Different Bacterial Species.

RNA synthetic biology tools have primarily been applied in E. coli ; however, many other bacteria are of industrial and clinical significance. Thus, the multicolor fluorogenic aptamer Pepper was evaluated in both Gram-positive and Gram-negative bacteria. Suitable HBC-Pepper dye pairs were identified that give blue, green, or red fluorescence signals in the E. coli , Bacillus subtilis , and Salmonella enterica serovar Typhimurium ( S . Typhimurium). Furthermore, we found that different RNA scaffolds have a drastic effect on in vivo fluorescence, which did not correlate with the in vitro folding efficiency. One such scaffold termed DF30-tRNA displays 199-fold greater fluorescence than the Pepper aptamer alone and permits simultaneous dual color imaging in live cells.