Ultra-Low-Cost RNA Sensors Using in-House Cell-Free Preparations

Implementation and use of RNA sensors in cell-free reactions in the global south is difficult due to the high cost of some reagents and the need for cold-chain shipping. In this work we optimized a low-cost protocol for the use of RNA toehold sensing with in-house cell-free reactions. We compared the expensive and cold-chain dependent 3-PGA energy source with the use of maltodextrin that can be transported at room temperature, finding higher end-point outputs with maltodextrin. Optimal magnesium concentration was defined for different batches, and the optimized extracts were compared with commercial kits such as PureXpress, and Promega S30 for the dynamics of production of GFP.

Our system was able to support sequence-specific RNA toehold sensing against portions of the Zika RNA using LacZ and LacZ-Alpha as outputs, obtaining better results using full length LacZ.

While plasmidic DNA is quite stable, linear DNA is prone to degradation in cell-free reactions based on crude extracts. Here we demonstrate how the stability of linear DNA can be increased in our extracts by suppressing specific nucleases with CRISPRi in cells before harvesting. In addition to an increase in linear DNA stability, these extracts had demonstrably increased protein production from linear DNA, as compared to plasmidic DNA.

We developed an in silico design strategy that leverages the NUPACK biophysical model of nucleotide sequences, which we have also made available for download as an open source repository called NupackSensors (http://www.github.com/elanibal/NupackSensors). Using NupackSensors, we screened four regions of the Potato Virus Y RNA, a virus that affects potato farmers in Chile, and selected a set of 16 different toehold RNA sensors for experimental screening. Our experimental results show how the optimized extracts can be used to quickly screen novel sensors.