DNA-based artificial molecular signaling system that mimics basic elements of reception and response

In order to maintain tissue homeostasis, cells communicate with the outside environment by receiving molecular signals, transmitting them, and responding accordingly with signaling pathways. Thus, one key challenge in engineering molecular signaling systems involves the design and construction of different modules into a rationally integrated system that mimics the cascade of molecular events. Herein, we rationally design a DNA-based artificial molecular signaling system that uses the confined microenvironment of a giant vesicle, derived from a living cell. This system consists of two main components. First, we build an adenosine triphosphate (ATP)-driven DNA nanogatekeeper. Second, we encapsulate a signaling network in the biomimetic vesicle, consisting of distinct modules, able to sequentially initiate a series of downstream reactions playing the roles of reception, transduction and response. Operationally, in the presence of ATP, nanogatekeeper switches from the closed to open state. The open state then triggers the sequential activation of confined downstream signaling modules.


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Weihong Tan
Dec 15, 2019 Fluorescence spectral data was measured on Fluoro Max-4 (Horiba Jobin Yvon). PAGE gel imaging data was collected using Microtek's Bio-6000 scanner and Bio-Rad ChemiDoc XRS System. Flow cytometry data was collected on the BD FACSVerse™ flow cytometer. Confocal imaging data was collected on Zeiss LSM 880 and Olympus FV1000 confocal laser scanning microscope.
Fluorescence spectral data was analyzed with OriginLab 2016. Statistical mean differences were evaluated using the unpaired Student's ttest with GraphPad 5. Flow cytometry data was analyzed using FlowJo 7.6.2. Confocal imaging data was analyzed using live softwares on Zeiss LSM 880 and Olympus FV1000 confocal laser scanning microscope.
The data that support the plots within this paper and other findings of this study are available from the corresponding author upon reasonable request.

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Life sciences study design
All studies must disclose on these points even when the disclosure is negative. The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.
Due to the high reproducibility and consistency between generating cell-mimicking giant vesicles from living cells, it was predetermined that a sample size of at least n=3 would allow for adequate analysis to reach meaningful conclusions of the data.
No data was excluded from studies.
All replication of experiments was successful; however, in the case of replication studies of AMSsys construction (Fig. 5), replication was not fully possible due to limited vesicles were survived after electroporation.
Throughout the whole experiment, samples were randomized into groups.
No blinding was used throughout experiments. All data collected was quantifiable and blinding would not change any bias in data collected.
HeLa and HepG2 cells were obtained from ATCC.
Cell cultures purchased from ATCC were authenticated by Short Tandem Repeat (STR) prior to purchase.
Cell lines were not tested for mycoplasma contamination.
No misidentified line for neither Hela cell nor HepG2 cell. Contamination of cells will not affect the generation of cellmimicking giant vesicles.