Abstract

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 temperat ure, 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 virus RNA using LacZ and LacZ-Alpha as outputs, obtaining better results using full length LacZ. The use of linear DNA as input for the cell-free reaction has several advantages respecting the use of plasmidic DNA as inputs for the reaction ( fast production using PCR amplification, no need for cloning in bacteria, etc ). Non-the-less it degraded easily in crude cell extracts. In this work, we demonstrate how the stability 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 two regions of the Potato Virus Y RNA, a virus that affects potato farmers in Chile, and selected a set of 8 different toehold RNA sensors for experimental screening. Our experimental results show that the optimized extracts can be used to quickly screen novel sensors, as they are fastlly produced by PCR.