As we have seen in the previous section, the tc aptamer has been used in several works together with the theophylline aptamer. The tetracycline aptamer consists of three stems (P1–P3), two single-stranded regions (the bulges, B1-2 and B2-3), and the loops L2 and L3 (see b). Portions of both bulges and the L3 loop are involved in tc binding
[42] that takes place at a very high affinity (K
D equals 0.8 nM
[43]). In 2003, Suess et al.
[44] reported the application of a tetracycline-binding aptamer to the control of gene expression in
S. cerevisiae. Green fluorescence was measured after placing the tc-responsive riboswitch in the 5′UTR of the strong
ADH1 promoter (pADH1), either close to the fluorescent protein start codon or in a cap-proximal location. The latter position appeared more effective in repression of translation (6-fold in the presence of tetracycline). Interestingly, stabilization of the aptamer structure led to an increased ON-to-OFF ratio but lowered basal fluorescence expression (i.e., in the absence of tetracycline). In another work published in 2003, the tetracycline riboswitch was shown to lead, in the presence of the antibiotic, to a 9-fold downregulation in luciferase synthesis from the constitutive
TEF1 promoter
[45]. 5′UTRs containing a variable number (one to three) of tetracycline aptamers were analyzed in
[46]. Initially, each configuration was placed between the
ADH1 promoter and the
GFP gene. As already experienced in
[44][45], riboswitches lowered the cell fluorescence level considerably when already in the absence of tetracycline. However, in the presence of 250 µM of the chemical, two and three aptamers caused an almost complete loss of fluorescence (1.1% and 0.6% of the fluorescence produced by pADH1 without aptamers) with a 21-fold and 37-fold repression factor compared to tetracycline-untreated cells. These riboswitch-containing 5′UTRs, preceded by either pADH1 or the
GPD promoter, were then adopted to construct universal insertion cassettes that could be integrated, via homologous recombination, into the yeast genome. Five endogenous genes (NEP1, NOP8, NOP14, PGI1, and SEC1) were completely switched OFF upon induction with at most 500 µM of tetracycline. NOP8 and SEC1 had never been regulated previously. More recently, the tandem configuration of this riboswitch was optimized in silico via machine learning techniques. In vivo, the newly designed 5′UTR sequence guaranteed a 40-fold repression of fluorescence in the presence of 250 µM tetracycline
[42].
One or two tetracycline-responsive riboswitches were also shown to be effective in controlling pre-mRNA splicing when placed within an intron nearby the 5′ splice site (SS). The most efficient regulation was obtained by burying the 5′ SS into the P1 stem at the basis of the tetracycline aptamer. Upon insertion of the modified intron along the sequence of GFP, fluorescence was emitted by the cells only in the absence of tetracycline, i.e., when the mRNA was spliced successfully. In contrast, in the presence of tetracycline a drastic reduction in cell fluorescence level was observed
[47].
In a different approach to control gene expression, the tetracycline aptamer was fused to the full-length HHR N79 from
Schistosoma mansoni via a linker region optimized through 11 rounds of in vitro selection (SELEX). Synthetic ribozymes completely self-cleaved in the presence of 1 µM of tetracycline, whereas self-cleavage was inhibited up to 333-fold in the absence of the antibiotic
[48].