Engineering a toolbox of synthetic dual-input hybrid riboswitches

The design of genetic regulators for the precise control of genes in complex genetic circuits remains a challenging task. Systems that rely on protein-based regulation to precisely control individual genes can suffer from the increased metabolic burden on the host cell organism that results from the additional gene expression required to produce the transcription factors. The introduction of longer genetic sequences required for the implementation of additional transcription factors (and their respective promoters/operators) can also be time consuming and difficult to adapt to a different host cell organism strain (codon optimization, difficulties during the cloning process of longer sequences, etc.). Synthetic riboswitches are a type of genetic element that can be used as an alternative to traditional protein-based regulation. Riboswitches are RNA sequences that form complex tertiary structures with the ability to specifically bind a designated target molecule (ligand) and regulate gene expression through a variety of different mechanisms. They are composed entirely of RNA, are compact in size (typically less than 100 nucleotides) and do not require additional factors to regulate gene expression. For these reasons, synthetic riboswitches have a low metabolic footprint, are strain independent, and can be easily adapted to different genetic circuits. While riboswitches were originally discovered in bacterial mRNAs, synthetic riboswitches have been engineered to function in all domains of life. Riboswitches are highly affine for their specific ligand, reaching picomolar binding affinities. A method called SELEX (Synthetic Evolution of Ligands by EXponential enrichment) can be used to discover new binding domains for any (water-soluble) target molecule of choice. An interesting type of riboswitch-based regulation in yeast is the roadblock mechanism. Regulation relies solely on a riboswitch, placed in the 5' UTR of a gene of interest, to stabilize its structure upon ligand binding to inhibit translation initiation by serving as a physical "roadblock" to the scanning ribosome. Since no sequestration of a regulatory sequence motif (e.g., ribosome binding site) is required, insertion of the riboswitch sequence into any 5' UTR is sufficient to enable regulation. A disadvantage of this type of regulation is that riboswitches exist in a pre-structured state even in the absence of ligand. As a result, the expression of the regulated gene is inhibited to some extent by the riboswitch even in the unbound state (basal expression). While previous studies have shown that the dynamic range (ratio of expression levels between the OFF and ON states) of these riboswitches can be increased by inserting multiple constructs in close spatial proximity (tandem riboswitches), the simultaneous increase of basal expression levels and dynamic range remains a challenge. A new type of dual-input riboswitch architecture, termed hybrid riboswitches, aims to solve this problem by incorporating two different ligand binding pockets into a single continuous structure. Hybrid riboswitches can rely on fewer scaffold sequences that would inhibit gene expression in the unbound state to provide stability, because the presence of the second binding pocket provides additional stabilization in the ligand-bound state. The idea was that hybrid riboswitches would retain the advantages of single-input riboswitches as compact and low-footprint regulators of gene expression, while providing the improved regulation of tandem riboswitches. This study aimed to create a variety of hybrid constructs from the combination of different parental riboswitches using both rational design and screening approaches. The goal was to create a toolbox of new highly efficient genetic regulators for use in circuit design, as well as to investigate how hybrid riboswitches can be efficiently designed. Several hybrid riboswitch constructs were generated that were able to outperform their parental riboswitches, with the best constructs achieving more than 50-60% basal expression and more than 20-fold dynamic range in the presence of both ligands. The best regulatory active construct achieved over 36-fold dynamic range in the presence of both ligands. In addition, it was investigated if the dual input nature of hybrid riboswitches could be used to design genetic regulators capable of emulating Boolean logic gate behavior. In collaboration with Heinz Koeppl's group, a screening pipeline was developed to discover a hybrid riboswitch responsive to neomycin and tetracycline that regulates gene expression only in the simultaneous presence of both ligands, thereby acting as a Boolean NAND gate.