Large-Conductance Transmembrane Porin Made from DNA Origami

DNA nanotechnology allows for the creation of three-dimensional structures at nanometer scale. Here, we use DNA to build the largest synthetic pore in a lipid membrane to date, approaching the dimensions of the nuclear pore complex and increasing the pore-area and the conductance 10-fold compared to previous man-made channels. In our design, 19 cholesterol tags anchor a megadalton funnel-shaped DNA origami porin in a lipid bilayer membrane. Confocal imaging and ionic current recordings reveal spontaneous insertion of the DNA porin into the lipid membrane, creating a transmembrane pore of tens of nanosiemens conductance. All-atom molecular dynamics simulations characterize the conductance mechanism at the atomic level and independently confirm the DNA porins’ large ionic conductance.

[1]  D. Case,et al.  Optimized particle-mesh Ewald/multiple-time step integration for molecular dynamics simulations , 2001 .

[2]  J O Bustamante,et al.  Electrical dimension of the nuclear envelope. , 2001, Physiological reviews.

[3]  R. Larson,et al.  The MARTINI Coarse-Grained Force Field: Extension to Proteins. , 2008, Journal of chemical theory and computation.

[4]  L. Toro,et al.  A neuronal beta subunit (KCNMB4) makes the large conductance, voltage- and Ca2+-activated K+ channel resistant to charybdotoxin and iberiotoxin. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[5]  P. E. Granum,et al.  Formation of Very Large Conductance Channels by Bacillus cereus Nhe in Vero and GH4 Cells Identifies NheA + B as the Inherent Pore-Forming Structure , 2010, The Journal of Membrane Biology.

[6]  Jeffery B. Klauda,et al.  Lipid chain branching at the iso- and anteiso-positions in complex Chlamydia membranes: a molecular dynamics study. , 2011, Biochimica et biophysica acta.

[7]  B. Brooks,et al.  Constant pressure molecular dynamics simulation: The Langevin piston method , 1995 .

[8]  Silvia Hernández-Ainsa,et al.  DNA origami nanopores for controlling DNA translocation. , 2013, ACS nano.

[9]  S. Howorka,et al.  A biomimetic DNA-based channel for the ligand-controlled transport of charged molecular cargo across a biological membrane. , 2016, Nature nanotechnology.

[10]  D. Tieleman,et al.  Molecular simulation of rapid translocation of cholesterol, diacylglycerol, and ceramide in model raft and nonraft membranes[S] , 2012, Journal of Lipid Research.

[11]  U. Keyser,et al.  Ion Channels Made from a Single Membrane-Spanning DNA Duplex , 2016, Nano letters.

[12]  U. Keyser,et al.  Lipid nanobilayers to host biological nanopores for DNA translocations. , 2013, Langmuir : the ACS journal of surfaces and colloids.

[13]  A. Aksimentiev,et al.  Molecular Dynamics of Membrane-Spanning DNA Channels: Conductance Mechanism, Electro-Osmotic Transport, and Mechanical Gating. , 2015, The journal of physical chemistry letters.

[14]  Robert D. Skeel,et al.  Correcting mesh-based force calculations to conserve both energy and momentum in molecular dynamics simulations , 2007, J. Comput. Phys..

[15]  P. Kollman,et al.  Settle: An analytical version of the SHAKE and RATTLE algorithm for rigid water models , 1992 .

[16]  Stefan Howorka,et al.  Bilayer-Spanning DNA Nanopores with Voltage-Switching between Open and Closed State , 2014, ACS nano.

[17]  Jejoong Yoo,et al.  Ionic conductivity, structural deformation, and programmable anisotropy of DNA origami in electric field. , 2015, ACS nano.

[18]  Nicholas A W Bell,et al.  DNA origami nanopores. , 2012, Nano letters.

[19]  D. O. Rudin,et al.  Reconstitution of Cell Membrane Structure in vitro and its Transformation into an Excitable System , 1962, Nature.

[20]  Vivek V. Thacker,et al.  Lipid-Bilayer-Spanning DNA Nanopores with a Bifunctional Porphyrin Anchor , 2013, Angewandte Chemie.

[21]  H. C. Andersen.  Rattle: A “velocity” version of the shake algorithm for molecular dynamics calculations , 1983 .

[22]  T. G. Martin,et al.  Synthetic Lipid Membrane Channels Formed by Designed DNA Nanostructures , 2012, Science.

[23]  U. Keyser,et al.  Protein reconstitution into freestanding planar lipid membranes for electrophysiological characterization , 2014, Nature Protocols.

[24]  Ulrich Koert,et al.  Synthetic ion channels. , 2004, Bioorganic & medicinal chemistry.

[25]  Laxmikant V. Kalé,et al.  Scalable molecular dynamics with NAMD , 2005, J. Comput. Chem..

[26]  A. Aksimentiev.  Deciphering ionic current signatures of DNA transport through a nanopore. , 2010, Nanoscale.

[27]  Adam H. Marblestone,et al.  Rapid prototyping of 3D DNA-origami shapes with caDNAno , 2009, Nucleic acids research.

[28]  E. Lindahl,et al.  3D pressure field in lipid membranes and membrane-protein complexes. , 2009, Physical review letters.

[29]  G. Patriarche,et al.  Biomimetic Nanotubes Based on Cyclodextrins for Ion-Channel Applications. , 2015, Nano letters.

[30]  K. Schulten,et al.  Microscopic Kinetics of DNA Translocation through synthetic nanopores. , 2004, Biophysical journal.

[31]  P. Schwille,et al.  DNA Nanostructures on Membranes as Tools for Synthetic Biology , 2016, Biophysical journal.

[32]  Jejoong Yoo,et al.  Water Mediates Recognition of DNA Sequence via Ionic Current Blockade in a Biological Nanopore. , 2016, ACS nano.

[33]  M. Klein,et al.  Constant pressure molecular dynamics algorithms , 1994 .

[34]  P. Rothemund.  Folding DNA to create nanoscale shapes and patterns , 2006, Nature.

[35]  Tim Liedl,et al.  DNA-Tile Structures Induce Ionic Currents through Lipid Membranes. , 2015, Nano letters.

[36]  Jejoong Yoo,et al.  Improved Parametrization of Li+, Na+, K+, and Mg2+ Ions for All-Atom Molecular Dynamics Simulations of Nucleic Acid Systems , 2012 .

[37]  Jejoong Yoo,et al.  De novo reconstruction of DNA origami structures through atomistic molecular dynamics simulation , 2016, Nucleic acids research.

[38]  Alexander D. MacKerell,et al.  All-atom empirical potential for molecular modeling and dynamics studies of proteins. , 1998, The journal of physical chemistry. B.

[39]  S. Howorka,et al.  Self-assembled DNA nanopores that span lipid bilayers. , 2013, Nano letters.

[40]  I. Tabushi,et al.  A,B,D,F-tetrasubstituted β-cyclodextrin as artificial channel compound , 1982 .

[41]  G. G. Stokes.  "J." , 1890, The New Yale Book of Quotations.

[42]  Jejoong Yoo,et al.  In situ structure and dynamics of DNA origami determined through molecular dynamics simulations , 2013, Proceedings of the National Academy of Sciences.

[43]  Carsten Kutzner,et al.  GROMACS 4:  Algorithms for Highly Efficient, Load-Balanced, and Scalable Molecular Simulation. , 2008, Journal of chemical theory and computation.

[44]  R. Benz.  Biophysical properties of porin pores from mitochondrial outer membrane of eukaryotic cells , 1990, Experientia.

[45]  K. Schulten,et al.  Imaging alpha-hemolysin with molecular dynamics: ionic conductance, osmotic permeability, and the electrostatic potential map. , 2005, Biophysical journal.

[46]  Jejoong Yoo,et al.  A comparison of coarse-grained and continuum models for membrane bending in lipid bilayer fusion pores. , 2013, Biophysical journal.