Abstract
Synthetic biologists have applied biomolecular engineering approaches toward the goal of novel biological devices and have shown progress in diverse areas of medicine and biotechnology. Especially promising is the application of synthetic biological devices towards a novel class of molecular diagnostics. As an example, a de-novo-designed riboregulator called toehold switch, with its programmability and compatibility with field-deployable devices showed promising in vitro applications for viral RNA detection such as Zika and Corona viruses. However, the in vivo application of high-performance RNA sensors remains challenging due to the secondary structure of long mRNA species. Here, we introduced ‘Helper RNAs’ that can enhance the functionality of toehold switch sensors by mitigating the effect of secondary structures around a target site. By employing the helper RNAs, previously reported mCherry mRNA sensor showed improved fold-changes in vivo. To further generalize the Helper RNA approaches, we employed automatic design pipeline for toehold sensors that target the essential genes within the pks island, an important target of biomedical research in connection with colorectal cancer. The toehold switch sensors showed fold-changes upon the expression of full-length mRNAs that apparently depended sensitively on the identity of the gene as well as the predicted local structure within the target region of the mRNA. Still, the helper RNAs could improve the performance of toehold switch sensors in many instances, with up to 10-fold improvement over no helper cases. These results suggest that the helper RNA approaches can further assist the design of functional RNA devices in vivo with the aid of the streamlined automatic design software developed here. Further, our solutions for screening and stabilizing single-stranded region of mRNA may find use in other in vivo mRNA-sensing applications such as cas13 crRNA design, transcriptome engineering, and trans-cleaving ribozymes.
Highlights
Synthetic biology is a burgeoning field that aims to design novel biological components, networks, and organisms by combining biological knowledge and technology with engineering principles [1,2]
Toehold switch sensors for detecting mCherry mRNA were previously characterized [19], where the presence of mCherry mRNA inputs leads to the unwinding of hairpin structure around the ribosome binding site (RBS) and start codon such that the downstream GFP output can be translated
Performance of helper RNAs was evaluated in the E. coli BL21 AI strain, where genomically encoded T7 RNA polymerase was induced by arabinose
Summary
Synthetic biology is a burgeoning field that aims to design novel biological components, networks, and organisms by combining biological knowledge and technology with engineering principles [1,2]. The underlying limitations of natural and engineered biological circuit components, such as undefined compatibility, low dynamic range, poor predictability, and crosstalk, make it challenging to realize the level of sophisticated synthetic biological designs that will drive future innovations. RNA-based synthetic gene regulatory components have an advantage that RNARNA interaction can be predicted via Watson–Crick base pairing. Synthetic biologists have endeavored to devise a novel riboregulator that controls transcription and/or translation in response to cognate RNAs [16,17,18]. Toehold switches are de-novo-designed riboregulators that regulate translation initiation of a downstream gene by sequestering the ribosome binding site (RBS) and starting codon [19] (Figure 1a)
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
