Applicability of a computational design approach for synthetic riboswitches

Abstract

Riboswitches have gained attention as tools for synthetic biology, since they enable researchers to reprogram cells to sense and respond to exogenous molecules. In vitro evolutionary approaches produced numerous RNA aptamers that bind such small ligands, but their conversion into functional riboswitches remains difficult. We previously developed a computational approach for the design of synthetic theophylline riboswitches based on secondary structure prediction. These riboswitches have been constructed to regulate ligand-dependent transcription termination in Escherichia coli. Here, we test the usability of this design strategy by applying the approach to tetracycline and streptomycin aptamers. The resulting tetracycline riboswitches exhibit robust regulatory properties in vivo. Tandem fusions of these riboswitches with theophylline riboswitches represent logic gates responding to two different input signals. In contrast, the conversion of the streptomycin aptamer into functional riboswitches appears to be difficult. Investigations of the underlying aptamer secondary structure revealed differences between in silico prediction and structure probing. We conclude that only aptamers adopting the minimal free energy (MFE) structure are suitable targets for construction of synthetic riboswitches with design approaches based on equilibrium thermodynamics of RNA structures. Further improvements in the design strategy are required to implement aptamer structures not corresponding to the calculated MFE state.

Figure 1.

Synthetic riboswitches for regulation of transcription termination in Escherichia coli based on de novo design. (A) Design principle. In the absence of ligand, the 3΄-part of the aptamer domain (red) forms a terminator hairpin with the reverse complementary part (blue), leading to transcription termination. Binding of the corresponding ligand (full black circle) stabilizes the aptamer conformation and disrupts the terminator structure, enabling transcription of the reporter gene. (B) Tetracycline-dependent riboswitch (RS) candidates from secondary structure predictions. Sequences include the tetracycline aptamer ‘cb32sh’ in red (40), a connecting spacer region in cyan, the complementary part for the aptamer in blue and a black U-stretch. Given below each sequence are the terminator and aptamer conformations in dot-bracket annotation (grey and red bars, respectively). Calculated free energy values of the competing structures and the terminator elements are indicated. (C) Riboswitch candidates for the streptomycin aptamer ‘motif 1’ (35), including predicted free energy values. Color code is according to (B).

Figure 2.

Activity tests of synthetic tetracycline riboswitches. (A) Design and activity of in silico predicted constructs. In the presence of 2.5 μg/ml tetracycline, expression of β-galactosidase was induced in E. coli, resulting in an increase of Miller units (MU). bgaBU₈ represents the appropriate positive control (19,33), consisting of a plasmid expressing the reporter gene under the same promoter but lacking the aptamer and the terminator hairpin. Only the U stretch is present, as this can have an effect on the reporter gene expression. (B) Optimized Tet-RS2 constructs. The terminator hairpin stability of Tet-RS2 was modified by deleting residues from the 3΄-end of the hairpin. In the activity test, these modifications led to an increased response ratio for Tet-RS2-17, Tet-RS2-15 and Tet-RS2-13, while in Tet-RS2-10, the terminator hairpin is too unstable to compete with the aptamer fold. Accordingly, the construct is in a constitutive ON state, independent of tetracycline binding. Furthermore, this behavior is a direct indication that the riboswitch constructs regulate at the level of transcription (19). (C) Serial arrangements of two and three Tet-RS2-15 repeats increase the ligand-dependent responsiveness. The existence of two or three intrinsic terminator elements leads to a reduction of background gene expression in the absence of tetracycline. While the monomeric Tet-RS2-15 shows an absolute activation of 80 MU that is not further increased by a higher tetracycline concentration, tandem and tridem show a somewhat lower absolute response (60–80 MU), with some increase at the higher ligand concentration. As a result, the activation ratios for the tandem and tridem are considerably increased compared to the monomeric riboswitch form.

Figure 3.

Design and activity test of synthetic streptomycin riboswitches. Measurement of β-galactosidase activity in MU was performed in the presence (1 mg/ml) or absence of streptomycin. While constructs Strep-RS 1, 4 and 8 showed no ligand-dependent response, Strep-RS7 exhibited a ligand-dependent 1.5-fold increase in gene expression. bgaBU₈: positive control without riboswitch (see legend Figure 2).

Figure 4.

Secondary structure analysis of the 5΄-labeled streptomycin aptamer transcript. (A) In-line probing analysis. T1: Partial digest with RNase T1. OH⁻: Alkaline hydrolysis. 0 h: Transcript without incubation. Lanes 0–10: Transcript was probed in the presence of indicated streptomycin concentrations for 44 h. Regions of high cleavage activity are indicated by brackets. (B) The secondary structure according to probing results is highly similar to streptomycin aptamer ‘motif 1’ (35) and differs from the in silico predicted structure of minimal free energy (MFE). (C) From the folding predictions of RNAsubopt (36), a slightly less stable structure with a free energy of −11.0 kcal/mol is highly similar to the probed one, indicating that the approach used the wrong fold for riboswitch design. Regions of high cleavage activity from structure probing in (A) are depicted as grey circles.

Figure 5.

Design principle and activity test of tandem riboswitches. The individual components Tet-RS2-15 and Theo-RS10 show a highly specific activation only in the presence of their cognate ligand. This is not affected by the presence of the second ligand. In the absence of ligands, both tandem riboswitches TheoTet-RS and TetTheo-RS show a minimal activation of β-galactosidase expression. In the presence of theophylline, the order of the riboswitch modules has an impact on background activity, indicating that in TetTheo-RS, the Tet module is affected in its OFF state, leading to a rather high background activation.