Expanding the toolbox of synthetic riboswitches with guanine-dependent aptazymes

Abstract

Artificial riboswitches based on ribozymes serve as versatile tools for ligand-dependent gene expression regulation. Advantages of these so-called aptazymes are their modular architecture and the comparably little coding space they require. A variety of aptamer-ribozyme combinations were constructed in the past 20 years and the resulting aptazymes were applied in diverse contexts in prokaryotic and eukaryotic systems. Most in vivo functional aptazymes are OFF-switches, while ON-switches are more advantageous regarding potential applications in e.g. gene therapy vectors. We developed new ON-switching aptazymes in the model organism Escherichia coli and in mammalian cell culture using the intensely studied guanine-sensing xpt aptamer. Utilizing a high-throughput screening based on fluorescence-activated cell sorting in bacteria we identified up to 9.2-fold ON-switches and OFF-switches with a dynamic range up to 32.7-fold. For constructing ON-switches in HeLa cells, we used a rational design approach based on existing tetracycline-sensitive ON-switches. We discovered that communication modules responding to tetracycline are also functional in the context of guanine aptazymes, demonstrating a high degree of modularity. Here, guanine-responsive ON-switches with a four-fold dynamic range were designed. Summarizing, we introduce a series of novel guanine-dependent ribozyme switches operative in bacteria and human cell culture that significantly broaden the existing toolbox.

Keywords: aptazyme; engineered riboswitch; hammerhead ribozyme; regulation of gene expression; synthetic biology.

Figure 1.

Screening and characterization of guanine-sensing aptazymes in E. coli. (a) A Type I HHR (black) is inserted in the 5′ UTR of an eGFP reporter gene. The Shine-Dalgarno (SD, blue) sequence is included in an extended stem I of the ribozyme. The xpt aptamer (light grey) is connected to the HHR via a communication module (green) with either four or six randomized nucleotides. (b) Bulk measurement of selected switches. The communication module of each switch is depicted underneath. An active HHR without aptamer domain served as control. Experiments were performed in triplicates and error bars represent standard deviation. (c) Dose dependency of the switches. The eGFP expression under control of the ON-switches Gua_eco1 and Gua_eco2 or the OFF-switches Gua_eco3 and Gua_eco4 relative to the expression under control of a HHRa is shown under varying guanosine concentrations. A sigmoidal fit was used to calculate EC50-values. Experiments were performed in triplicates and error bars represent standard deviation. (d) Flow cytometry histograms of the eGFP expression under control of the ON-switches Gua_eco1 and Gua_eco2 or OFF-switches Gua_eco3 and Gua_eco4 in absence (black) and presence (grey) of 1 mM guanosine.

Figure 2.

Cleavage activities of aptazyme sequences in vitro. Percentage of cleaved fraction for ON-switches (a) Gua_eco1, (b) Gua_eco2 and OFF-switches (c) Gua_eco3, (d) Gua_eco4 in absence (black line) and presence (grey dashed lines) of guanine.

Figure 3.

Rational design of guanine-sensing aptazymes in HeLa cells. (a) The tetracycline aptamer in the TetK4/K19 constructs (10) is replaced by the guanine-sensing xpt aptamer (16), while the respective communication module (green) remains identical. Additional tested communication modules (K3, K4.1, K9, K18) are presented as well. Nucleotides involved in the stem I-stem II tertiary interactions of the ribozyme are encircled in red. A ribozyme inactivating A→G mutation is depicted. (b) Dual luciferase assay of the constructs shown in (a). Transfected cells were incubated in the absence or presence of 500 µM guanosine. Experiments were performed in triplicates and error bars represent standard deviation. A psiCheck2 plasmid without aptazyme serves as control. (c) Dose dependency of the Gua_K3, Gua_K4.1, Gua_K9 and Gua_K18 switches. The relative luciferase activity under control of the respective switch and an inactivated variant is shown under varying guanosine concentrations. A sigmoidal fit was used to calculate the EC50-value. Experiments were performed in triplicates and error bars represent standard deviation.