(429d) Effectiveness of Nuclease Inhibition for Stabilizing Linear DNA in Cell-Free Expression Systems

Cell-free expression (CFE) systems are powerful tools used for applications ranging from environmental sensors to point-of-use diagnostics due to their quick reaction times, affordability, and portability. CFE allows the use of linear DNA templates, which have benefits over plasmids (the typical form of DNA used in CFE) because they shorten the prototyping cycle to a few hours by eliminating the days it takes to clone and replicate plasmids in vivo. However, linear DNA added to a CFE system is degraded by endogenous nucleases present in the lysate. This can be detrimental for the implementation of prominent linear DNA-based technologies like aptamers as CFE sensors. Here, we assess the effectiveness of multiple strategies to stabilize two types of linear DNA in CFE systems: PCR-generated gene templates and DNA aptamers. These strategies include use of the competitive inhibitor GamS, non-canonical DNA backbones, longer DNA templates, and oligos encoding Chi sites (short DNA motifs that signal the RecBCD nuclease), some of which have previously been shown to inhibit nucleases from degrading linear DNA templates for gene expression. We found that even with Chi DNA, linear templates with strong synthetic σ⁷⁰ promoters expressed less than 1% of the protein produced from a phage T7 promoter, while the inducible P_BAD promoter could not produce detectable protein levels. We also showed that high levels of Chi DNA are unable to stabilize DNA aptamers long enough for them to be used effectively as sensors in CFE systems. These results indicate that the level of protein expressed from linear templates depends heavily upon immediate downstream amplification steps, and the same nuclease inhibition strategies cannot extend aptamer half-life enough for proper functionality. Continuing to explore the limits of nuclease inhibition is critical for enhancing the use of linear DNA in CFE systems and could enable the creation of impactful new sensor platforms.