Short Activators and Repressors of RNA Toehold Switches.

RNA toehold switches are a widely used class of molecule to detect specific RNA "trigger" sequences, but their design, intended function, and characterization to date leave it unclear whether they can function properly with triggers shorter than 36 nucleotides. Here, we explore the feasibility of using standard toehold switches with 23-nucleotide truncated triggers. We assess the crosstalk of different triggers with significant homology and identify a highly sensitive trigger region where just one mutation from the consensus trigger sequence can reduce switch activation by 98.6%. However, we also find that triggers with as many as seven mutations outside of this region can still lead to 5-fold induction of the switch. We also present a new approach using 18- to 22-nucleotide triggers as translational repressors for toehold switches and assess the off-target regulation for this strategy as well. The development and characterization of these strategies could help enable applications like microRNA sensors, where well-characterized crosstalk between sensors and detection of short target sequences are critical.

Corrigendum: Effective use of linear DNA in cell-free expression systems.

[This corrects the article DOI: 10.3389/fbioe.2021.715328.].

Effective Use of Linear DNA in Cell-Free Expression Systems.

Cell-free expression systems (CFEs) are cutting-edge research tools used in the investigation of biological phenomena and the engineering of novel biotechnologies. While CFEs have many benefits over in vivo protein synthesis, one particularly significant advantage is that CFEs allow for gene expression from both plasmid DNA and linear expression templates (LETs). This is an important and impactful advantage because functional LETs can be efficiently synthesized in vitro in a few hours without transformation and cloning, thus expediting genetic circuit prototyping and allowing expression of toxic genes that would be difficult to clone through standard approaches. However, native nucleases present in the crude bacterial lysate (the basis for the most affordable form of CFEs) quickly degrade LETs and limit expression yield. Motivated by the significant benefits of using LETs in lieu of plasmid templates, numerous methods to enhance their stability in lysate-based CFEs have been developed. This review describes approaches to LET stabilization used in CFEs, summarizes the advancements that have come from using LETs with these methods, and identifies future applications and development goals that are likely to be impactful to the field. Collectively, continued improvement of LET-based expression and other linear DNA tools in CFEs will help drive scientific discovery and enable a wide range of applications, from diagnostics to synthetic biology research tools.