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Published March 26, 2023 | Published + Supplemental Material
Journal Article Open

Digital nanoreactors to control absolute stoichiometry and spatiotemporal behavior of DNA receptors within lipid bilayers


Interactions between membrane proteins are essential for cell survival but are often poorly understood. Even the biologically functional ratio of components within a multi-subunit membrane complex—the native stoichiometry—is difficult to establish. Here we demonstrate 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 (modelling membrane proteins). 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, 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 Author(s) 2023. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. 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 the National Institute of Mental Health awards MH125320 (to P.W.K.R. and E.R.C.), MH061876 and NS097362 (to E.R.C.), and by the National Science Foundation awards CCF 2106695 (to C.T.), a Faculty Early Career Development Award CCF 2143227 (to C.T.) and a Future Manufacturing award MCB 2134772 (to P.W.K.R). E.R.C. is an investigator at 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. Data availability: Data in Figs. 2 and 3, Supplementary Figures 3–6, 8, and 10, and DNA receptor sequences in Table 1 and Supplementary Table 1 are provided in the Source Data file. Chemicals catalogs are provided as Supplementary Data 1 and DNA staple sequences are provided as Supplementary Data 2. Source data are provided with this paper. Code availability: Files related to DNA origami design (see folder cadnano), CRN simulator (see folder crn), and PERL scripts (see folder perl_scripts) are provided as Supplementary Software files. Contributions: V.M. conceived the original idea, designed, performed, and analyzed most experiments, and wrote the manuscript's 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. modeled the DNA circuit. E.R.C. mentored and hosted V.M. All authors discussed the results and participated in manuscript writing. Competing interests: Certain aspects related to the internal immobilization and the release of membrane receptors employing DNA origami are covered in a pending US provisional patent application (63/415,546) owned by the California Institute of Technology with inventor V.M. The other authors declare no competing interests.

Attached Files

Published - s41467-023-36996-x.pdf

Supplemental Material - 41467_2023_36996_MOESM1_ESM.pdf

Supplemental Material - 41467_2023_36996_MOESM2_ESM.pdf

Supplemental Material - 41467_2023_36996_MOESM3_ESM.xlsx

Supplemental Material - 41467_2023_36996_MOESM4_ESM.xlsx

Supplemental Material - 41467_2023_36996_MOESM5_ESM.zip

Supplemental Material - 41467_2023_36996_MOESM7_ESM.xlsx


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August 22, 2023
October 20, 2023