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Published March 28, 2023 | Submitted + Supplemental Material
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Digital nanoreactors for control over absolute stoichiometry and spatiotemporal behavior of receptors within lipid bilayers


Interactions between membrane proteins are essential for cell survival and proper function, but the structural and mechanistic details of these interactions are often poorly understood. Even the biologically functional ratio of protein components within a multi-subunit membrane complex—the native stoichiometry—is difficult to establish. We have demonstrated digital nanoreactors that can control interactions between lipid-bound molecular receptors along three key dimensions: stoichiometric, spatial, and temporal. Each nanoreactor is based on a DNA origami ring, which both templates the synthesis of a liposome and provides tethering sites for DNA-based receptors. Receptors are released into the liposomal membrane using strand displacement and a DNA logic gate measures receptor heterodimer formation. High-efficiency tethering of receptors enables the kinetics of receptors in 1:1 and 2:2 absolute stoichiometries to be observed by bulk fluorescence in a plate reader which in principle is generalizable to any ratio. Similar 'single molecule in bulk' experiments using DNA-linked membrane proteins could determine native stoichiometry and the kinetics of membrane protein interactions for applications ranging from signalling research to drug discovery.

Additional Information

The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. V.M. acknowledges Erik Winfree and Lulu Qian for both their comments and access to equipment for initial experiments. V.M. thanks Human Frontier Science Program (HFSP LT001164/2017-L) for a Postdoctoral Fellowship and Caltech for additional support. This study was supported by National Institute of Mental Health awards MH125320 (to P.W.K.R. and E.R.C.), MH061876 and NS097362 (to E.R.C.), and a Faculty Early Career Development Award from NSF CCF 2143227 (to C.T.). E.R.C. is an investigator of the Howard Hughes Medical Institute (HHMI). This article is subject to HHMI's Open Access to Publications policy. HHMI lab heads have previously granted a nonexclusive CC BY 4.0 license to the public and a sublicensable license to HHMI in their research articles. Pursuant to those licenses, the author-accepted manuscript of this article can be made freely available under a CC BY 4.0 license immediately upon publication. Author contributions. V.M. conceived the original idea, designed, performed and analyzed most experiments, and wrote the manuscript first draft. V.M., Z.Z., C.T. and P.W.K.R. contributed further ideas. C.T., V.M., and P.W.K.R. designed the DNA logic circuit and analyzed kinetics data. Z.Z. designed the origami, assisted in DOL synthesis, and performed TEM. N.S. and C.T. modelled the DNA circuit. E.R.C. mentored and hosted V.M. All authors discussed the results and participated in manuscript writing. The authors have declared no competing interest.

Attached Files

Submitted - 2022.10.04.509789v1.full.pdf

Supplemental Material - media-1.pdf

Supplemental Material - media-2.zip


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Additional details

August 20, 2023
October 18, 2023