Functional Nanopores Enabled with DNA

Membrane-spanning nanopores are used in label-free single-molecule sensing and next-generation portable nucleic acid sequencing, and as powerful research tools in biology, biophysics, and synthetic biology. Naturally occurring protein and peptide pores, as well as synthetic inorganic nanopores, are used in these applications, with their limitations. The structural and functional repertoire of nanopores can be considerably expanded by functionalising existing pores with DNA strands and by creating an entirely new class of nanopores with DNA nanotechnology. This review outlines progress in this area of functional DNA nanopores and outlines developments to open up new applications

Functional Nanopores Enabled with DNA

Membrane-spanning nanopores are used in label-free single-molecule sensing and next-generation portable nucleic acid sequencing, and as powerful research tools in biology, biophysics, and synthetic biology. Naturally occurring protein and peptide pores, as well as synthetic inorganic nanopores, are used in these applications, with their limitations. The structural and functional repertoire of nanopores can be considerably expanded by functionalising existing pores with DNA strands and by creating an entirely new class of nanopores with DNA nanotechnology. This review outlines progress in this area of functional DNA nanopores and outlines developments to open up new applications

A Light-Triggered Synthetic Nanopore for Controlling Molecular Transport Across Biological Membranes

Controlling biological molecular processes with light is of high interest in biological research and biomedicine, as light allows precise and selective activation in a non-invasive and non-toxic manner. A molecular process benefitting from light control is the transport of cargo across biological membranes, which is conventionally achieved by membrane-puncturing barrel-shaped nanopores. Yet, there is considerable interest to construct more complex gated pores. Here, we pioneer a synthetic light-gated nanostructure which controls transport across membranes via a controllable lid. The light-triggered nanopore is self-assembled from six pore DNA strands and a lid strand carrying light-switchable azobenzene molecules. Exposure to light opens the pore to allow small-molecule transport across membranes. Our light-triggered pore advances biomimetic chemistry and DNA nanotechnology and may be used in biotechnology, biosensing, targeted drug release, or synthetic cells

Molecular Recognition in Confined Space Elucidated with DNA Nanopores and Single-Molecule Force Microscopy

The binding of ligands to receptors within a nanoscale small space is relevant in biology, biosensing, and affinity filtration. Binding in confinement can be studied with biological systems but under the limitation that essential parameters cannot be easily controlled including receptor type and position within the confinement and its dimensions. Here we study molecular recognition with a synthetic confined nanopore with controllable pore dimension and molecular DNA receptors at different depth positions within the channel. Binding of a complementary DNA strand is studied at the single-molecule level with atomic force microscopy. Following the analysis, kinetic association rates are lower for receptors positioned deeper inside the pore lumen while dissociation is faster and requires less force. The phenomena are explained by the steric constraints on molecular interactions in confinement. Our study is the first to explore recognition in DNA nanostructures with atomic force microscopy and lays out new tools to further quantify the effect of nanoconfinement on molecular interactions

DNA nanostructures for membrane probing and controlled poration

DNA nanotechnology excels at self-assembling structurally defined functional nanodevices. A recent avenue is to develop nanostructures that act at the bilayer interface to investigate or control membrane properties. The goal of this PhD is to create novel DNA nanostructures that probe and puncture lipid bilayers. Probing these bilayer properties is important for biophysical research and also for developing a diagnostic tool for various diseases such as cancer where circulating cancer cells express altered membrane elasticity compared to healthy cells. Currently, elasticity can be measured by tools such as atomic force microscopy (AFM) which is serial and requires specialised equipment. In comparison, the thesis describes a novel nanodevice built out of DNA, named DNA nanoactuator (NA), which binds to membranes and upon addition of single DNA strands deforms those membranes. The NA offers a highly parallel and simultaneous non-invasive analysis via fluorescence readout and broadens access to the nanodevice for any user of fluorescence microscopy. Aside from membrane probing, this PhD project features a DNA valve for controllable membrane poration. Nanochannels are the most relevant border crossings in nature but their application is restricted due to the complicated introduction of wide-ranging modifications in their structure and function. A broadened structural range can be attained with DNA as building materials for nanochannels. To expand their function, a light trigger for controlled opening and closing and tuneable membrane transport was constructed. Photoswitchable azobenzene molecules were incorporated into the DNA nanochannel to control duplex dissociation for channel opening and closing. The light-gated pore allows precise design, quick assembly, and easy, non-invasive activation. The pore can find applications in artificial cell systems and for targeted drug delivery. In conclusion, the proposed DNA nanostructures advance the field of DNA nanotechnology and present new routes to understand membranes or alter their permeation properties for basic and applied science