Aptamers, Riboswitches, and Ribozymes in S. cerevisiae Synthetic Biology

Abstract

Among noncoding RNA sequences, riboswitches and ribozymes have attracted the attention of the synthetic biology community as circuit components for translation regulation. When fused to aptamer sequences, ribozymes and riboswitches are enabled to interact with chemicals. Therefore, protein synthesis can be controlled at the mRNA level without the need for transcription factors. Potentially, the use of chemical-responsive ribozymes/riboswitches would drastically simplify the design of genetic circuits. In this review, we describe synthetic RNA structures that have been used so far in the yeast Saccharomyces cerevisiae. We present their interaction mode with different chemicals (e.g., theophylline and antibiotics) or proteins (such as the RNase III) and their recent employment into clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas) systems. Particular attention is paid, throughout the whole paper, to their usage and performance into synthetic gene circuits.

Keywords: S. cerevisiae; aptamers; riboswitches; ribozymes; synthetic biology.

Figure 1

The theophylline aptamer. (a) Theophylline structure (PubChem CID 2153). (b) The secondary structure of the theophylline aptamer. (c) The theophylline-responsive antiswitch as a means to control gene expression [32]. The RNA sequence complementary to the target green fluorescence protein (GFP) transcript is highlighted in red. (d) Fusion of a theophylline aptamer to the stem II of a hammerhead ribozyme. The resulting aptazyme undergoes autocleavage in the presence of 5 µM of theophylline causing an over 15-fold decrease in fluorescence expression. (e) Theophylline-induced translational frame shift. Here, the theophylline aptamer is fused to a ‒1 programmed ribosomal frameshifting (PRF) RNA structure [37].

Figure 2

Antibiotic-responsive aptamers and the ribozyme-gRNA-ribozyme (RGR) cassette. (a) Tetracycline structure (PubChem CID 54675776). (b) The tetracycline aptamer. (c) Neomycin structure (PubChem CID 8378). (d) The neomycin aptamer. The main structural features of the two RNA molecules are here highlighted. (e) RGR cassette for the expression of single guide RNAs. Upon autocleavage of the HH and the hepatitis delta virus (HDV) ribozymes, the single guide RNA is released. sgRNA binds Cas9 and brings it to the target DNA that is finally cut. The green line denotes the spacer, whereas the gray hairpin represents the direct repeat.

Figure 3

Rnt1p substrate. (a) The hairpin cleaved by the Rnt1 protein consisted of three main parts: The IBPB (initial binding and positioning box); the BSB (binding stability box); and the CEB (cleavage efficiency box). The “Clamp” is a spacer sequence needed to insulate and stabilize the hairpin [92]. (b) Theophylline aptamer fused to the Rnt1p hairpin substrate in the GFP 3′UTR. In the presence of theophylline, the secondary structure of the Rnt1p substrate is modified, which inhibits the cleavage by Rnt1p.