Split aminoacyl-tRNA synthetases for proximity-induced stop codon suppression

Abstract

Synthetic biology tools for regulating gene expression have many useful biotechnology and therapeutic applications. Most tools developed for this purpose control gene expression at the level of transcription, and relatively few methods are available for regulating gene expression at the translational level. Here, we design and engineer split orthogonal aminoacyl-tRNA synthetases (o-aaRS) as unique tools to control gene translation in bacteria and mammalian cells. Using chemically induced dimerization domains, we developed split o-aaRSs that mediate gene expression by conditionally suppressing stop codons in the presence of the small molecules rapamycin and abscisic acid. By activating o-aaRSs, these molecular switches induce stop codon suppression, and in their absence stop codon suppression is turned off. We demonstrate, in Escherichia coli and in human cells, that split o-aaRSs function as genetically encoded AND gates where stop codon suppression is controlled by two distinct molecular inputs. In addition, we show that split o-aaRSs can be used as versatile biosensors to detect therapeutically relevant protein–protein interactions, including those involved in cancer, and those that mediate severe acute respiratory syndrome-coronavirus-2 infection.

Impact of exogenous aminoacyl-tRNA synthetase and tRNA on temperature sensitivity inEscherichia coli

“…First, we have introduced a novel ncAA responsive module into the biological logic gate, expanding the scope of external stimuli, in addition to the rare examples. 38, 39 Second, we showcased a new cold-inducible system, addressing potential issues with the stress effects and physiological responses associated with heat shock. 42 Lastly, unlike previous systems vulnerable to mutations (e.g., strategies with simple amber codon insertion), our system remains robust due to its absolute dependency on BzF for dimer formation.…”

“…The development of biological logic gates for precise control of gene expression remains an ongoing challenge. Various synthetic modules for circuit design have been explored, 28 including the use of amber suppressors, 36 orthogonal ribosome/orthogonal mRNA pairs, 37 genetic code expansion, 38, 39 and small RNAs. 40 Achieving this control relies on the development of genetic circuits that can effectively respond to externally applicable stimuli while maintaining compatibility with one another.…”

Split Proteins and Reassembly Modules for Biological Applications

“…The variant of Methanocaldococcus jannaschii tyrosyl‐tRNA synthetase (MjTyrRS) was also reported to be split at 99/100 and 268/269, identifying these as the most efficient split sites among the 5 regions. These split MaPylRS and MjTyrRS were then applied in the detection of PPIs and the construction of ncAA‐sensitive biological logic gates [1a] . Such techniques could be further adapted to create additional splits in various orthogonal aaRSs, enhancing the array of tools for ncAA‐responsive biological circuitry.…”

“…These synthetic variants are particularly valuable in split protein reassembly applications due to their minimal interference with host protein functions and their ability to form stable, specific non‐covalent interactions. Examples include split site screening and validation with sTetR‐SYNZIP17/18 fusions, [38–39] and the validation of split sites in biological logic gate construction utilizing split PylRS [1a] or split MHT (Figure 4). [16a] …”

“…For instance, a logic gate was designed using split‐T7RNAP and a temperature‐sensitive TlpA binding pair to modulate gene expression and cell growth via temperature changes [1d] . Another study introduced a split aaRS‐based logic gate that controls stop codon suppression, showcasing the potential utilities of using ncAAs as synthetic inputs [1a] …”