Correlation of mRNA delivery timing and protein expression in lipid-based transfection

A. Reiser; D. Woschée; N. Mehrotra; R. Krzyszton; H. H. Strey; J. O. Rädler

The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/607986 doi: bioRxiv preprint molecules m0, which are simultaneously delivered at a defined time t0. The time t0 represents the time when translation begins and we refer to it as the onset time. The model assumes that all mRNA molecules are delivered at the same time, which clearly is a simplification. However, the time interval during which mRNA is released into the cytosol is short compared to the time scale of expression and, therefore, can be treated as a single event in time. Defining the time point of incubation as zero, the expression onset time is the period from incubation until beginning of translation, which includes all intermediated processes such as cellular uptake, release into the cytosol and unpacking of the complexes. The analytical solution to the ODEs is given in the Materials and Methods section and were used to fit the measured single-cell fluorescence time courses using least-square fitting (21). To prevent overfitting the data by using too many free parameters of the translation-maturation model we fixed the maturation rate kM and the protein degradation rate β to the mean population values. These mean values were determined in an independent translation block experiment using cycloheximide performed within a previous study (22) . The fitting results of the model with the fixed parameters are in good agreement with the experimental data. Figure 2B shows representative single-cell time courses with the respective fits. The magnification of the representative data illustrates that the expression onset times (black dots author/funder. All rights reserved. No reuse allowed without permission.