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1.D.46 The DNA Nanopore (DnaNP) Family

DNA Nanopores (DNPs) consist of artificial biological channels embedded in cell membranes that regulate ionic transport by responding to external stimuli such as pH, voltage, and molecular binding and could theoretically be involved in biosensing, drug delivery and ionic circuit construction (Harrell et al. 2004; Langecker et al. 2012; Langecker et al. 2014). Buchsbaum et al. 2014 described nanopores that can simultaneously respond to pH and transmembrane potential changes. DNA oligomers containing protonatable A and C bases are attached at the narrow opening of an asymmetric nanopore. Lowering the pH to 5.5 causes the positively charged DNA molecules to bind to other strands with negative backbones, thereby creating an electrostatic mesh that closes the pore to unprecedentedly high resistances of several tens of gigaohms. At neutral pH values, voltage switching causes the isolated DNA strands to undergo nanomechanical movement, as seen by a reversible current modulation. Buchsbaum et al. 2014 provided evidence that the pH-dependent reversible closing mechanism is robust and applicable to nanopores with inner pore diameters of up to 14 nm. The concept of creating an electrostatic mesh may be applied to other organic polymers. Some DNPs exhibit voltage switching between open (low voltage) and closed (high voltage) states (Seifert et al. 2014).  The main ion conducting path has been shown to run through the membrane-spanning channel lumen (Seifert et al. 2014).

Self-assembled DNA nanostructures have been used to create man-made transmembrane channels in lipid bilayers. A DNA-tile structure with a subnanometer channel and cholesterol-tags for membrane anchoring has an outer diameter of 5 nm and a molecular weight of 45 kDa, the dimensions of synthetic nanostructures comparable to biological ion channels (Göpfrich et al. 2015). Because of its simple design, the structure self-assembles within a minute, making its creation scalable for applications in biology. Ionic current recordings demonstrate that the tile structures enable ion conduction through lipid bilayers and show gating and voltage-switching behavior.  DNA-based membrane channels have openings much smaller than that of the archetypical six-helix bundle. Phosphorothioate-modified DNA nanopores are able to structurally mimic biological channels for molecular transport across live cell membranes (Lv et al. 2020). With its stable structure and small hollow size (<2 nm) as well as its ability to interact with the lipid molecules, this DNA nanopore could insert into cellular plasma membranes. This membrane-spanning channel could transport ions and antitumor drugs into neurons and cancer cells, respectively (Lv et al. 2020).

The structures of DNA channels and their conductance mechanisms were studied by Yoo and Aksimentiev 2015. They reported the results of molecular dynamics simulations that characterized the biophysical properties with atomic precision. While remaining stable, the local structures of the channels undergo considerable fluctuations, departing from the idealized design. The transmembrane ionic current flows both through the central pore of the channel as well as along the DNA walls and through the gaps in the DNA structure. They found that conductance depends on the membrane tension, making them suitable for force sensing applications. Electro-osmosis governs drug-like molecule transport (Aksimentiev 2015). 

Ion conduction can be induced by a single DNA duplex that lacks a hollow central channel. Decorated with six porpyrin-tags, the duplex is designed to span lipid membranes. Combining electrophysiology measurements with all-atom molecular dynamics simulations, Göpfrich et al. 2016 elucidated the microscopic conductance pathway. Ions flow at the DNA-lipid interface as the lipid head groups tilt toward the amphiphilic duplex forming a toroidal pore filled with water and ions. Ionic current traces produced by the DNA-lipid channel show well-defined insertion steps, closures, and gating similar to those observed for traditional protein channels or synthetic pores. Ionic conductances obtained through simulations and experiments are in excellent quantitative agreement. The conductance mechanism with the smallest possible DNA-based ion channel offers a route to design a new class of synthetic ion channels with maximal simplicity. 

Göpfrich et al. 2016 used 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 tenfold compared to previous man-made channels. In their design, nineteen cholesterol-tags anchor a megadalton funnel-shaped DNA origami porin in a lipid bilayer membrane. Confocal imaging and ionic current recordings revealed spontaneous insertion of the DNA porin into the lipid membrane, creating a transmembrane pore of tens of nanosiemens conductance. All-atom molecular dynamics simulations characterized the conductance mechanism at the atomic level and independently confirmed the DNA porins' large ionic conductance.  DNA nanopores are used as probes for sensing in addition to using substrates for sequencing (Liu and Wu 2016). 

DNA-based nanopores, synthetic biomolecular membrane pores whose geometry and chemical functionality can be tuned using the tools of DNA nanotechnology, make them promising molecular devices for applications in single-molecule biosensing and synthetic biology. Krishnan et al. 2016 introduced a large DNA membrane channel with an ≈4 nm diameter pore, which has stable electrical properties and spontaneously inserts into flat lipid bilayer membranes. Membrane incorporation is facilitated by a large number of hydrophobic functionalizations or, alternatively, streptavidin linkages between biotinylated channels and lipids. The channel displays an Ohmic conductance of ≈3 nS, consistent with its size, and allows electrically driven translocation of single-stranded and double-stranded DNA analytes. Using confocal microscopy and a dye influx assay, Krishnan et al. 2016 demonstrated the spontaneous formation of membrane pores in giant unilamellar vesicles. Pores can be created both in an outside-in and an inside-out configuration.  

Joshi and Maiti 2017 engineered synthetic DNA nanopores through lipid bilayer membranes to access the interior of a cell and demonstrated the stability and dynamics of a tile-based 6-helix DNA nanotube (DNT) embedded in a POPC lipid bilayer. The head groups of the lipids close to the lumen cooperatively tilt towards the hydrophilic sugar-phosphate backbone of the DNA and form a toroidal structure around the patch of DNT protruding in the membrane. Transmembrane ionic current measurements revealed the I-V characteristics of the water filled DNT lumen in the tested lipid membrane. Simulations of the DNTs with ssDNA and dsDNA overhangs at the mouth of the pore showed gating effects with differences in the transmembrane ionic conductivities for open and closed state nanopores (Joshi and Maiti 2017). Functional and biomimetic nanostructures on and in lipid membranes (Wu et al. 2018) as well as the characterization and applications of biomimetic DNA nanotubes (Liu et al. 2018) have been reviewed. 

A synthetic DNA nanopore can accommodate folded proteins. Transport of fluorescent proteins through single pores has been kinetically analysed to reveal an at least 20-fold higher speed for the electrically driven movement. These pores also allow a high diffusive flux of more than 66 molecules per second that can also be directed beyond equillibria (Diederichs et al. 2019). Vesicular fusogenic DNA nanopores can controllably open lipid membranes, depolarize the plasma membrane and induce apoptosis in cancer cells (Chen et al. 2019). 

Thomsen et al. 2019 used DNA origami to create a synthetic 9 nm wide DNA nanopore, controlled by programmable, lipidated flaps and equipped with a size-selective gating system for the translocation of macromolecules. Successful assembly and insertion of the nanopore into lipid bilayers were validated by transmission electron microscopy (TEM), while selective translocation of cargo and the pore mechanosensitivity were studied using optical methods, including single-molecule, total internal reflection fluorescence (TIRF) microscopy. Size-specific cargo translocation and oligonucleotide-triggered opening of the pore were demonstrated, showing that the DNA nanopore can function as a real-time detection system for external signals, offering potential for a variety of highly parallelized sensing applications (Thomsen et al. 2019).

References associated with 1.D.46 family:

Buchsbaum, S.F., G. Nguyen, S. Howorka, and Z.S. Siwy. (2014). DNA-modified polymer pores allow pH- and voltage-gated control of channel flux. J. Am. Chem. Soc. 136: 9902-9905. 24992159
Chen, L., S. Liang, Y. Chen, M. Wu, and Y. Zhang. (2019). Destructing Plasma Membrane with activatable vesicular DNA nanopores. ACS Appl Mater Interfaces. [Epub: Ahead of Print] 31815409
Diederichs, T., G. Pugh, A. Dorey, Y. Xing, J.R. Burns, Q. Hung Nguyen, M. Tornow, R. Tampé, and S. Howorka. (2019). Synthetic protein-conductive membrane nanopores built with DNA. Nat Commun 10: 5018. 31685824
Gopfrich K., Zettl T., Meijering AE., Hernandez-Ainsa S., Kocabey S., Liedl T. and Keyser UF. (2015). DNA-Tile Structures Induce Ionic Currents through Lipid Membranes. Nano Lett. 15(5):3134-8. 25816075
Göpfrich, K., C.Y. Li, I. Mames, S.P. Bhamidimarri, M. Ricci, J. Yoo, A. Mames, A. Ohmann, M. Winterhalter, E. Stulz, A. Aksimentiev, and U.F. Keyser. (2016). Ion Channels Made from a Single Membrane-Spanning DNA Duplex. Nano Lett. [Epub: Ahead of Print] 27324157
Göpfrich, K., C.Y. Li, M. Ricci, S.P. Bhamidimarri, J. Yoo, B. Gyenes, A. Ohmann, M. Winterhalter, A. Aksimentiev, and U.F. Keyser. (2016). Large-Conductance Transmembrane Porin Made from DNA Origami. ACS Nano. [Epub: Ahead of Print] 27504755
Harrell, C.C., P. Kohli, Z. Siwy, and C.R. Martin. (2004). DNA-nanotube artificial ion channels. J. Am. Chem. Soc. 126: 15646-15647. 15571378
Joshi, H. and P.K. Maiti. (2017). Structure and electrical properties of DNA nanotubes embedded in lipid bilayer membranes. Nucleic Acids Res. [Epub: Ahead of Print] 29136243
Krishnan, S., D. Ziegler, V. Arnaut, T.G. Martin, K. Kapsner, K. Henneberg, A.R. Bausch, H. Dietz, and F.C. Simmel. (2016). Molecular transport through large-diameter DNA nanopores. Nat Commun 7: 12787. 27658960
Langecker, M., V. Arnaut, J. List, and F.C. Simmel. (2014). DNA nanostructures interacting with lipid bilayer membranes. Acc Chem Res 47: 1807-1815. 24828105
Langecker, M., V. Arnaut, T.G. Martin, J. List, S. Renner, M. Mayer, H. Dietz, and F.C. Simmel. (2012). Synthetic lipid membrane channels formed by designed DNA nanostructures. Science 338: 932-936. 23161995
Liu, L. and H.C. Wu. (2016). DNA-Based Nanopore Sensing. Angew Chem Int Ed Engl. [Epub: Ahead of Print] 27676313
Liu, X., Y. Zhao, P. Liu, L. Wang, J. Lin, and C. Fan. (2018). Biomimetic DNA Nanotubes: Nanoscale Channel Design and Applications. Angew Chem Int Ed Engl. [Epub: Ahead of Print] 30290046
Lv, C., X. Gu, H. Li, Y. Zhao, D. Yang, W. Yu, D. Han, J. Li, and W. Tan. (2020). Molecular Transport through a Biomimetic DNA Channel on Live Cell Membranes. ACS Nano. [Epub: Ahead of Print] 32897687
Seifert A., Gopfrich K., Burns JR., Fertig N., Keyser UF. and Howorka S. (2015). Bilayer-spanning DNA nanopores with voltage-switching between open and closed state. ACS Nano. 9(2):1117-26. 25338165
Thomsen, R.P., M.G. Malle, A.H. Okholm, S. Krishnan, S.S. Bohr, R.S. Sørensen, O. Ries, S. Vogel, F.C. Simmel, N.S. Hatzakis, and J. Kjems. (2019). A large size-selective DNA nanopore with sensing applications. Nat Commun 10: 5655. 31827087
Wu, N., F. Chen, Y. Zhao, X. Yu, J. Wei, and Y. Zhao. (2018). Functional and Biomimetic DNA Nanostructures on Lipid Membranes. Langmuir. [Epub: Ahead of Print] 30044097
Yoo J. and Aksimentiev A. (2015). Molecular Dynamics of Membrane-Spanning DNA Channels: Conductance Mechanism, Electro-Osmotic Transport, and Mechanical Gating. J Phys Chem Lett. 6(23):4680-7. 26551518