Sorting sub-150-nm liposomes of distinct sizes by dna-brick-assisted centrifugation

ABSTRACT In cells, myriad membrane-interacting proteins generate and maintain curved membrane domains with radii of curvature around or below 50 nm. To understand how such highly curved

membranes modulate specific protein functions, and vice versa, it is imperative to use small liposomes with precisely defined attributes as model membranes. Here, we report a versatile and

scalable sorting technique that uses cholesterol-modified DNA ‘nanobricks’ to differentiate hetero-sized liposomes by their buoyant densities. This method separates milligrams of liposomes,

regardless of their origins and chemical compositions, into six to eight homogeneous populations with mean diameters of 30–130 nm. We show that these uniform, leak-resistant liposomes serve

as ideal substrates to study, with an unprecedented resolution, how membrane curvature influences peripheral (ATG3) and integral (SNARE) membrane protein activities. Compared with

conventional methods, our sorting technique represents a streamlined process to achieve superior liposome size uniformity, which benefits research in membrane biology and the development of

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SIMILAR CONTENT BEING VIEWED BY OTHERS SINGLE-PARTICLE COMBINATORIAL MULTIPLEXED LIPOSOME FUSION MEDIATED BY DNA Article 04 April 2022 UNRAVELING THE SURFACE MARKER SIGNATURE OF CELL-DERIVED

VESICLES VIA PROTEOME ANALYSIS AND NANOPARTICLE FLOW CYTOMETRY Article Open access 02 January 2024 A DNA-NANOASSEMBLY-BASED APPROACH TO MAP MEMBRANE PROTEIN NANOENVIRONMENTS Article 02

November 2020 DATA AVAILABILITY Source data are provided with this paper. The data (TEM images, gel and blot images, fluorescence traces and statistical data) supporting the findings of this

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work is supported by a National Institutes of Health (NIH) Director’s New Innovator Award (GM114830), an NIH grant (GM132114) and a Yale University faculty startup fund to C.L., NIH grants

to E.R.C. (MH061876 and NS097362), to T.M. (GM100930 and GM109466) and to E.K. (NS113236), and a National Key Research and Development Program of China grant (2020YFA0908901) and National

Natural Science Foundation of China grants (21673050, 91859104 and 81861138004) to H.G. Author E.R.C. is an Investigator of the Howard Hughes Medical Institute. Q.X. is supported by a

Graduate Scholarship from the Agency for Science, Technology and Research (Singapore). AUTHOR INFORMATION Author notes * These authors contributed equally: Yang Yang, Zhenyong Wu. AUTHORS

AND AFFILIATIONS * Department of Cell Biology, Yale University School of Medicine, New Haven, CT, USA Yang Yang, Laurie Wang, Qiancheng Xiong, Longfei Liu, Thomas J. Melia & Chenxiang

Lin * Nanobiology Institute, Yale University, West Haven, CT, USA Yang Yang, Zhenyong Wu, Qiancheng Xiong, Longfei Liu, Erdem Karatekin & Chenxiang Lin * Institute of Molecular Medicine,

Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China Yang Yang * Department of Cellular and Molecular Physiology, Yale University School of Medicine, New

Haven, CT, USA Zhenyong Wu & Erdem Karatekin * Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA Kaifeng Zhou, Yong Xiong & Erdem Karatekin *

Institutes of Biomedical Sciences, Fudan University, Shanghai, China Kai Xia & Hongzhou Gu * Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital, Fudan University,

Shanghai, China Kai Xia & Hongzhou Gu * Howard Hughes Medical Institute, Department of Neuroscience, University of Wisconsin-Madison, Madison, WI, USA Zhao Zhang & Edwin R. Chapman *

Saints-Pères Paris Institute for the Neurosciences (SPPIN), Centre National de la Recherche Scientifique (CNRS) UMR 8003, Université de Paris, Paris, France Erdem Karatekin Authors * Yang

Yang View author publications You can also search for this author inPubMed Google Scholar * Zhenyong Wu View author publications You can also search for this author inPubMed Google Scholar *

Laurie Wang View author publications You can also search for this author inPubMed Google Scholar * Kaifeng Zhou View author publications You can also search for this author inPubMed Google

Scholar * Kai Xia View author publications You can also search for this author inPubMed Google Scholar * Qiancheng Xiong View author publications You can also search for this author inPubMed

 Google Scholar * Longfei Liu View author publications You can also search for this author inPubMed Google Scholar * Zhao Zhang View author publications You can also search for this author

inPubMed Google Scholar * Edwin R. Chapman View author publications You can also search for this author inPubMed Google Scholar * Yong Xiong View author publications You can also search for

this author inPubMed Google Scholar * Thomas J. Melia View author publications You can also search for this author inPubMed Google Scholar * Erdem Karatekin View author publications You can

also search for this author inPubMed Google Scholar * Hongzhou Gu View author publications You can also search for this author inPubMed Google Scholar * Chenxiang Lin View author

publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS Y.Y. initiated the project, designed and performed most of the experiments, analysed the data and

prepared the manuscript. Z.W. performed the membrane fusion study and analysed the data. L.W. performed the lipidation study. K.Z. performed the cryo-EM study. K.X. replicated the sorting

method. Q.X., L.L. and Z.Z. performed the negative-stain TEM study. Y.X. supervised the cryo-EM study and interpreted the data. T.J.M. designed and supervised the lipidation study and

interpreted the data. E.K. and E.R.C. supervised the membrane fusion study and interpreted the data. H.G. designed the liposome leakage assay, supervised replication of the sorting method

and interpreted the data. C.L. initiated the project, designed and supervised the study, interpreted the data and prepared the manuscript. All authors participated in the discussions, and

reviewed and approved the manuscript. CORRESPONDING AUTHORS Correspondence to Hongzhou Gu or Chenxiang Lin. ETHICS DECLARATIONS COMPETING INTERESTS Yale University has filed a provisional

patent (US Application No. 62/968,683; inventors: C.L. and Y.Y.) on the DNA-assisted liposome-sorting method. ADDITIONAL INFORMATION PEER REVIEW INFORMATION _Nature Chemistry_ thanks the

anonymous reviewers for their contribution to the peer review of this work. PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims in published maps and

institutional affiliations. SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION Supplementary Tables 1–5, Figs. 1–28 and Notes 1 and 2. REPORTING SUMMARY SOURCE DATA SOURCE DATA FIG. 1

Statistical source data of liposome diameters. SOURCE DATA FIG. 2 Unprocessed gels. SOURCE DATA FIG. 3 Unprocessed gels and western blots. SOURCE DATA FIG. 4 Statistical source data of

liposome diameters, raw data of fluorescence traces and data underlying plots. RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Yang, Y., Wu, Z., Wang, L.

_et al._ Sorting sub-150-nm liposomes of distinct sizes by DNA-brick-assisted centrifugation. _Nat. Chem._ 13, 335–342 (2021). https://doi.org/10.1038/s41557-021-00667-5 Download citation *

Received: 03 February 2020 * Accepted: 23 February 2021 * Published: 30 March 2021 * Issue Date: April 2021 * DOI: https://doi.org/10.1038/s41557-021-00667-5 SHARE THIS ARTICLE Anyone you

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