Synthetic biology has advanced the design of standardized control devices that program cellular functions and metabolic activities in living organisms. Rational interconnection of these synthetic switches resulted in increasingly complex designer networks that execute input-triggered genetic instructions with precision, robustness and computational logic reminiscent of electronic circuits. Using trigger-controlled transcription factors, which independently control gene expression, and RNA-binding proteins that inhibit the translation of transcripts harbouring specific RNA target motifs, we have designed a set of synthetic transcription–translation control devices that could be rewired in a plug-and-play manner. Here we show that these combinatorial circuits integrated a two-molecule input and performed digital computations with NOT, AND, NAND and N-IMPLY expression logic in single mammalian cells. Functional interconnection of two N-IMPLY variants resulted in bitwise intracellular XOR operations, and a combinatorial arrangement of three logic gates enabled independent cells to perform programmable half-subtractor and half-adder calculations. Individual mammalian cells capable of executing basic molecular arithmetic functions isolated or coordinated to metabolic activities in a predictable, precise and robust manner may provide new treatment strategies and bio-electronic interfaces in future gene-based and cell-based therapies.

Switch‐hitter: A new strategy to control gene expression relies on a ligand‐dependent, ribozyme‐based RNA switch. The key feature of this setup is that the ribozyme liberates the ribosome binding site upon cleavage of the message. The design made it possible to screen in vivo for an inducible aptazyme by attaching an aptamer to stem III of the ribozyme, thus preserving naturally occurring stem I/stem II contacts.

RNA-based modules such as riboswitches represent straightforward and simplified approaches for the regulation of gene expression, as no additional proteins are needed. G-rich sequences are known to adopt stable four-stranded structures, and such quadruplexes have been suspected to play important roles in key functions such as the control of gene expression. Here we demonstrate that RNA quadruplexes readily form in vivo. We have constructed mRNA-based G-rich elements that mask the ribosome binding site by folding into four-stranded structures. The suppression of gene expression correlates with the stability of inserted G quadruplexes. Moreover, quadruplexes with moderate stability respond to changes in temperature, thus behaving as artificial RNA thermometers. In conclusion, we introduce tuneable mRNA-based devices that enable modulation of gene expression by a predictable but thus far unknown mechanism.

Four-stranded DNA and RNA quadruplexes or G4 motifs are non-B DNA conformations that are presumed to form in vivo, although only few explicit evidence has been reported. Using bioinformatics the presence of putative DNA G-quadruplexes within critical promoter regions has been demonstrated and a regulatory role in transcription has been suspected. However, in genomic DNA the presence of the complementary strand interferes with the potential to form a quadruplex motif. Contrarily RNA G4 motifs have no such limitation and consequently strong interference with gene expression is suspected. Nevertheless, experimental evidence is scarce. Here we show a well-defined structure–function relationship of synthetic quadruplex sequences in 5′-UTRs in multiple mammalian cell-lines. We establish a universal ‘translational suppressor’ effect of these motifs on gene expression at the translational level and show for the first time that specific features such as loop-length and the number of ‘GGG’-repeats further determine the suppressive impact. Moreover, a consistent and predictable repression of gene expression is observed for naturally occurring RNA G4 motifs, augmenting the functional relevance of these unusual nucleic acid structures.