Programming cell interactions with light using oligonucleotides

Introduction::
The complex arrangement of cells within tissue leads to the formation of cell junctions between different cells types (heterojunctions), which play an important role in regulating tissue and biological functional and dysfunctional behaviors that include gene expression levels, cell migration, and mitochondrial transfer. Given the important role of contact-mediated cellular communication, technologies spanning lithography, microfluidics, and three-dimension (3D) printing have been developed to enable control over 3D cell placement for probing fundamental biological processes, printing tissues, and creating disease models. To enable cell assembly with high precision, the programmability and specificity of DNA complementary base pairing has been used for printing cellular structures with higher-resolution spatial control. Specifically, DNA mediated interactions were used to program the assembly of cells to create 3D structures consisting of multiple different cell types and have been recently been leveraged to create defined cell interactions on material substrates.^1-4 While exquisite control has been achieved with nucleic acid-based cell assembly, on demand spatial control in cell compatible conditions remains limited. In response, we have utilized photocaged DNA, strands containing nucleotides that can be activated with light, on cell surfaces to program cell-cell interactions with non-UV wavelengths ( >400 nm). By combining photocaged DNA coated cells with a digital-micromirror-device (DMD), we programmed cell interaction in defined locations on cell monolayers.




Materials and Methods::
DNA was synthesized containing standard bases along with photocaged coumarin protected guanine (5'-Dimethoxytrityl-N2-(4-isopropylphenoxyacetyl)-O6-[[7-(diethylamino)coumarin-4-yl]-methyl]-2'-deoxyGuanosine, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite) on an Expedite synthesizer following manufacturer recommendations. Deprotection was performed using water saturated with ammonium hydroxide (28-32%; Fisher Chemicals; 24 h room temperature) and purified using a Glen-Pac DNA purification column (Glen Research). For attaching DNA to cells, an amino-containing oligonucleotide was included, and the DNA was incubated with 10x concentrated transcyclooctene (TCO)- N-Hydroxysuccinimide (NHS) ester to create a TCO-terminated DNA. M4A4-GFP cells were incubated with sulfo-NHS-ester-PEG4-methyltetrazine (MTET) at 1-10 mM concentrations for 5 mins to convert amines on the cell surface to MTET. TCO terminated oligonucleotides were then incubated with MTET modified cells to attach the DNA. M4A4 cells were grown into high confluency and incubated with 1 mM MTET-Sulfo-NHS in 150 mM NaCl and 10 mM HEPES saline (pH 8.4) for 5 minutes at 30°C. The cells were rinsed with 1X PBS, then incubated with 100 µM DNA containing 1-5 photocaged bases. The cells were placed under a microscope that had a DMD (Mightex) mounted on it for controlling light and exposed to 425 nm light for 60 seconds. The cells were rinsed with 1X PBS and then incubated with either cells functionalized with complementary DNA or with Cy5-labeled complementary DNA. After 5 minutes, the samples were rinsed with PBS and imaged.




Results, Conclusions, and Discussions:: To assess ability to activate photocaged DNA on the surface of cells, M4A4 cells were functionalized with DNA containing 1, 3, or 5 photocaged bases. The photocaged DNA-coated cells were incubated with Cy5 fluorophore-labeled complementary DNA with or without light exposure. Flow cytometry assessment of cell fluorescence shows that DNA with 5 photocaged oligonucleotides prevented hybridization with Cy5 complementary DNA until exposed to light, while 1 or 3 modifications did prevent hybridization (Figure A). Building off our ability to control DNA recognition on cell surfaces with light, we evaluated whether we could control cell assembly. When cells coated with photocaged oligonucleotides were incubated with cells coated with complementary DNA, only cells exposed to 425 nm light assembled into larger structures (Figure B).

To evaluate performance of photocaged DNA on cell monolayers with spatial control, we attached DNA containing 5 photocaged bases to the surface of cell monolayers. The monolayers were then exposed to light controlled by a DMD for fixed periods of time ranging from 1 s to 60 s. Upon incubation with complementary Cy5 labeled DNA, fluorescence was confined to the defined regions. Enrichment occurred at exposure times as short as 1 s with no significant increases in fluorescence observed at times less than 30 s (Figure C). Using the same DMD setup, we demonstrate spatially controlled cell placement on monolayers. M4A4-GFP cell monolayers were functionalized with photocaged DNA. Cells were then exposed to light in the shape of a star and then incubated with complementary DNA-coated cells. Upon rinsing, we observed cell enrichment reflecting the defined pattern (Figure D).

Collectively, our results demonstrate a pathway to creating complex cell assemblies with high resolution spatial control using DNA. Importantly, the use of photocaged oligonucleotides is rapid and easily controlled with low-photoxicity wavelengths ( >400 nm). Given the ability to create unique DNA sequences with programmable recognition, our technology can be extended to include multiplexed printing of more complex cell clusters with multiple layers. Overall, this strategy opens a viable pathway to creating defined and uniform cell structures for drug discovery and tissue engineering applications.




Acknowledgements (Optional): : Research reported in this abstract was supported by NIGMS of the National Institutes of Health under award number R21GM141563. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

References (Optional): :
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4 Prahl, L. S., Porter, C. M., Liu, J., Viola, J. M. & Hughes, A. J. Independent control over cell patterning and adhesion on hydrogel substrates for tissue interface mechanobiology. iScience2022).