Canonical translation-modulating OFF-riboswitches with a single aptamer binding to a small molecule that function in a higher eukaryotic cell-free expression system

Cell-free riboswitches

“…Nonetheless, the variety of ligands that cell-free riboswitches have been engineered to detect are quite broad compared to cell-based riboswitches. Although theophylline has been, by far, the most popular trigger molecule, cell-free riboswitches that respond to biotin, 77 tetramethylrhodamine (TMR), 46,78,96 flavin mononcleotide (FMN), 88,89,97 cGMP, 36,87 TPP, 39,42 fluoride, 48 histamine, 45 dopamine, 46 thyroxine, 46 sulphorhodamine B, 88 Hoechst dye 33258, 76 tobramycin, 76 tetracycline, 88,97 and pentadeoxyribonucleotides 114 have been designed (Tables 1 and 2). With the exception of histamine, however, these cell-free riboswitches have been constructed using known aptamers from natural riboswitches or discovered Fig.…”

“…2B ). 78 It is worth noting that similar riboswitch designs have been shown to function in yeast 79–82 and in mammalian cells. 76 …”

The Theophylline Aptamer: 25 Years as an Important Tool in Cellular Engineering Research

“…However, addition of theophylline to the wheat germ extract stabilized the 3′ CITE stem, leading to an over 7-fold increase in translation. In another study, they developed a simple riboswitch that functions in wheat germ extract by placing the aptamer upstream of the yellow fluorescent protein gene . They varied distance between the aptamer and gene as well as the length of the upper stem in the aptamer and obtained a maximum ON/OFF ratio of 5.9 when the aptamer was placed 85 nt upstream of the gene with a 7-nt upper stem.…”

“…In another study, they developed a simple riboswitch that functions in wheat germ extract by placing the aptamer upstream of the yellow fluorescent protein gene. 107 They varied distance between the aptamer and gene as well as the length of the upper stem in the aptamer and obtained a maximum ON/OFF ratio of 5.9 when the aptamer was placed 85 nt upstream of the gene with a 7-nt upper stem. The 85 nt spacer between aptamer and gene is much greater than that the 8−10 nt observed for maximal activity in bacteria, and provides another important data point to begin to design riboswitches de novo for use in eukaryotic systems.…”

In Vivo Validation of a Reversible Small Molecule-Based Switch for Synthetic Self-Amplifying mRNA Regulation

“…However, this concentration is above the toxic serum level of tetracycline in humans (i.e., 30 mg/L). 20 Similarly, a robust regulation of theophylline-responsive mRNAs has been obtained in cell-free expression systems, [21][22][23][24] bacteria, [25][26][27][28][29][30][31] yeast, 32 and mammalian cell lines 33,34 in the presence of 0.5-5 mM (i.e., 90.1-900.8 mg/L) theophylline. Again, this concentration is not achievable in humans, as theophylline is toxic at serum levels >0.111 mM (i.e., 20 mg/L).…”