TY - JOUR
T1 - Single molecule delivery into living cells
AU - Chau, Chalmers C.
AU - Maffeo, Christopher M.
AU - Aksimentiev, Aleksei
AU - Radford, Sheena E.
AU - Hewitt, Eric W.
AU - Actis, Paolo
N1 - We thank Prof Nikita Gamper of the University of Leeds for providing the DRG neurons. We thank Prof Derek Steele of the University of Leeds for providing technical support and access to the spinning disk confocal microscope. We thank Prof Joshua Edel for sharing the nanopore data analysis script with us. We thank Dr Michael Davies for providing the fluorescently labelled fibrils. C.C. and P.A. acknowledge funding from the Engineering and Physical Sciences Research Council UK (EPSRC) Healthcare Technologies for the grant EP/W004933/1. C.C. and P.A. acknowledge funding from the Biotechnology and Biological Sciences Research Council (BBSRC) for the Research Grant BB/X003086/1. E.H. and S.E.R. acknowledge funding from the Wellcome Trust for the Research Grant 094266/Z/10/Z.\u00A0S.E.R. holds a Royal Society Professorial Fellowship (RSRP/R1/211057). This work was supported by the Human Frontier Science Project (RGP0047/2020) and the Leverhulme Visiting Professorship grant [VP2-2019-012] to A.A. ARBD development is supported by the National Science Foundation grant OAC-2311550. The supercomputer time was provided through the Leadership Resource allocation MCB20012 on Frontera of the Texas Advanced Computing Center and the ACCESS allocation MCA05S028. For the purpose of Open Access, the authors have applied a CC BY public copyright licence to any author accepted manuscript version arising from this submission.
We thank Prof Nikita Gamper of the University of Leeds for providing the DRG neurons. We thank Prof Derek Steele of the University of Leeds for providing technical support and access to the spinning disk confocal microscope. We thank Prof Joshua Edel for sharing the nanopore data analysis script with us. We thank Dr Michael Davies for providing the fluorescently labelled fibrils. C.C. and P.A. acknowledge funding from the Engineering and Physical Sciences Research Council UK (EPSRC) Healthcare Technologies for the grant EP/W004933/1. C.C. and P.A. acknowledge funding from the Biotechnology and Biological Sciences Research Council (BBSRC) for the Research Grant BB/X003086/1. E.H. and S.E.R. acknowledge funding from the Wellcome Trust for the Research Grant 094266/Z/10/Z. S.E.R. holds a Royal Society Professorial Fellowship (RSRP/R1/211057). This work was supported by the Human Frontier Science Project (RGP0047/2020) and the Leverhulme Visiting Professorship grant [VP2-2019-012] to A.A. ARBD development is supported by the National Science Foundation grant OAC-2311550. The supercomputer time was provided through the Leadership Resource allocation MCB20012 on Frontera of the Texas Advanced Computing Center and the ACCESS allocation MCA05S028. For the purpose of Open Access, the authors have applied a CC BY public copyright licence to any author accepted manuscript version arising from this submission.
PY - 2024/12
Y1 - 2024/12
N2 - Controlled manipulation of cultured cells by delivery of exogenous macromolecules is a cornerstone of experimental biology. Here we describe a platform that uses nanopipettes to deliver defined numbers of macromolecules into cultured cell lines and primary cells at single molecule resolution. In the nanoinjection platform, the nanopipette is used as both a scanning ion conductance microscope (SICM) probe and an injection probe. The SICM is used to position the nanopipette above the cell surface before the nanopipette is inserted into the cell into a defined location and to a predefined depth. We demonstrate that the nanoinjection platform enables the quantitative delivery of DNA, globular proteins, and protein fibrils into cells with single molecule resolution and that delivery results in a phenotypic change in the cell that depends on the identity of the molecules introduced. Using experiments and computational modeling, we also show that macromolecular crowding in the cell increases the signal-to-noise ratio for the detection of translocation events, thus the cell itself enhances the detection of the molecules delivered.
AB - Controlled manipulation of cultured cells by delivery of exogenous macromolecules is a cornerstone of experimental biology. Here we describe a platform that uses nanopipettes to deliver defined numbers of macromolecules into cultured cell lines and primary cells at single molecule resolution. In the nanoinjection platform, the nanopipette is used as both a scanning ion conductance microscope (SICM) probe and an injection probe. The SICM is used to position the nanopipette above the cell surface before the nanopipette is inserted into the cell into a defined location and to a predefined depth. We demonstrate that the nanoinjection platform enables the quantitative delivery of DNA, globular proteins, and protein fibrils into cells with single molecule resolution and that delivery results in a phenotypic change in the cell that depends on the identity of the molecules introduced. Using experiments and computational modeling, we also show that macromolecular crowding in the cell increases the signal-to-noise ratio for the detection of translocation events, thus the cell itself enhances the detection of the molecules delivered.
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U2 - 10.1038/s41467-024-48608-3
DO - 10.1038/s41467-024-48608-3
M3 - Article
C2 - 38782907
AN - SCOPUS:85194129413
SN - 2041-1723
VL - 15
JO - Nature communications
JF - Nature communications
IS - 1
M1 - 4403
ER -