Spatiotemporal Patterning of Living Cells with Extracellular DNA Programs

Abstract

Reactive extracellular media focus on engineering reaction networks outside the cell to control intracellular chemical composition across time and space. However, current implementations lack the feedback loops and out-of-equilibrium molecular dynamics for encoding spatio-temporal control. Here, we demonstrate that enzyme-DNA molecular programs combining these qualities are functional in an extracellular medium where human cells can grow. With this approach, we construct an internalization program that delivers fluorescent DNA inside living cells and remains functional for at least 48 h. Its non-equilibrium dynamics allows us to control both the time and position of cell internalization. In particular, a spatially inhomogeneous version of this program generates a tunable reaction-diffusion two-band pattern of cell internalization. This demonstrates that a synthetic extracellular program can provide temporal and positional information to living cells, emulating archetypal mechanisms observed during embryo development.We foresee that non-equilibrium reactive extracellular media could be advantageously applied to in vitro biomolecular tracking, tissue engineering or smart bandages.

Long-lasting and responsive DNA/enzyme-based programs in serum-supplemented extracellular media

“…We have demonstrated that greater DTT concentrations improve the responsiveness of the DNA program in the presence of serum. However, since high levels of [DTT] decrease cell 10 viability, as they transiently activate endoplasmic reticulum stress 27 and cause cell detachment, 3 we decided to further increase the biocompatibility of our buer to mitigate cytotoxicity (although the introduction of FBS already partially attenuates the adverse eect of DTT, Figure S16). To do so, we increased the concentration of the cell culture medium (from 0.5x to 0.89x), we removed non-critical PEN buer components and lowered the concentration of magnesium (SI Section 3) to create a new buer that we named Cell+ buer.…”

“…Although we found an average ∼2-fold reduction in growth rate compared to the control, a ∼2-fold increase in viable cell number after 48 h was achieved compared to previously reported experimental conditions in the absence of FBS (Kin buer). 3 To stress the importance of developing serum-compatible DNA/enzyme programs, we studied the growth of human embryonic kidney 293 cells (HEK 293 cell line), as their behaviour (e.g. signalling pathways) is signicantly aected by serum starvation.…”

“…Images and data were treated as previously reported. 3 Briey, an ImageJ / Fiji (NIH) routine was implemented to stack the time-lapse images of each well. Subsequently, a Matlab routine was used to create an intensity prole across time of each well, which was divided by an initial time point to correct from inhomogeneous illumination between wells.…”

“…In an eort to surpass this limitation, we recently demonstrated the embedding of a DNA/enzyme molecular program within the extracellular medium of an in vitro cell culture. 3 Notably, we showed how the programmable extracellular medium was capable of guiding cellular composition across time and space, opening the pathway towards the development of extracellular synthetic biology tools. Nevertheless, the absence of serum reduces the biological signicance of that study, since the myriad of bioactive substances provided by the animal serum has become an essential component for successful in vitro cell culture.…”

“…In the last few decades, the programmability and reactivity of DNA has positioned the DNA nanotechnology eld as a promising avenue for the development of biomolecular pharmaceutical approaches. 1 In particular, DNA molecular programming tools have been extensively used to create bioactuation systems for the delivery of cargoes 2 and for the modication of cellular composition, 3 as well as biosensing tools for cell sorting 4 and molecular detection. 5 For example, the specic amplication of nucleic acid sequences by polymerase-based DNA programs allows the detection of microRNA biomarkers down to attomolar concentrations with dynamic ranges up to 10 orders of magnitude.…”