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

Nature Chemistry volume 13, pages 335–342 (2021)Cite this article

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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 liposomal drug-delivery systems.

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Fig. 1: DNA-brick-assisted liposome-sorting scheme and results.
Fig. 2: Sorting liposomes containing self-cleaving deoxyribozymes.
Fig. 3: ATG3-catalysed GL1 lipidation reaction studied using uniformly sized liposomes.
Fig. 4: SNARE-mediated membrane fusion studied using uniformly-sized liposomes.

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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 study are available within the paper and its Supplementary Information files.

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Acknowledgements

This 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

  1. These authors contributed equally: Yang Yang, Zhenyong Wu.

Authors and Affiliations

  1. 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

  2. Nanobiology Institute, Yale University, West Haven, CT, USA

    Yang Yang, Zhenyong Wu, Qiancheng Xiong, Longfei Liu, Erdem Karatekin & Chenxiang Lin

  3. Institute of Molecular Medicine, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China

    Yang Yang

  4. Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT, USA

    Zhenyong Wu & Erdem Karatekin

  5. Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA

    Kaifeng Zhou, Yong Xiong & Erdem Karatekin

  6. Institutes of Biomedical Sciences, Fudan University, Shanghai, China

    Kai Xia & Hongzhou Gu

  7. Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital, Fudan University, Shanghai, China

    Kai Xia & Hongzhou Gu

  8. Howard Hughes Medical Institute, Department of Neuroscience, University of Wisconsin-Madison, Madison, WI, USA

    Zhao Zhang & Edwin R. Chapman

  9. Saints-Pères Paris Institute for the Neurosciences (SPPIN), Centre National de la Recherche Scientifique (CNRS) UMR 8003, Université de Paris, Paris, France

    Erdem Karatekin

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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.

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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.

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Peer review information Nature Chemistry thanks the anonymous reviewers for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Tables 1–5, Figs. 1–28 and Notes 1 and 2.

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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.

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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

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