We created nanometer-scale transmembrane channels in lipid bilayers using self-assembled DNA-based nanostructures. Scaffolded DNA origami was used to create a stem that penetrates and spans a lipid membrane, and a barrel-shaped cap that adheres to the membrane in part via 26 cholesterol moieties. In single-channel electrophysiological measurements, we find similarities to the response of natural ion channels, such as conductances on the order of 1 nS and channel gating. More pronounced gating was seen for mutations in which a single DNA strand of the stem protruded into the channel. In single-molecule translocation experiments, we highlight one of many potential applications of the synthetic channels, namely as single DNA molecule sensing devices.

DNA-based nanopores are synthetic biomolecular membrane pores, whose geometry and chemical functionality can be tuned using the tools of DNA nanotechnology, making them promising molecular devices for applications in single-molecule biosensing and synthetic biology. Here we introduce 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, we demonstrate the spontaneous formation of membrane pores in giant unilamellar vesicles. Pores can be created both in an outside-in and an inside-out configuration.

CONSPECTUS: DNA has been previously shown to be useful as a material for the fabrication of static nanoscale objects, and also for the realization of dynamic molecular devices and machines. In many cases, nucleic acid assemblies directly mimic biological structures, for example, cytoskeletal filaments, enzyme scaffolds, or molecular motors, and many of the applications envisioned for such structures involve the study or imitation of biological processes, and even the interaction with living cells and organisms. An essential feature of biological systems is their elaborate structural organization and compartmentalization, and this most often involves membranous structures that are formed by dynamic assemblies of lipid molecules. Imitation of or interaction with biological systems using the tools of DNA nanotechnology thus ultimately and necessarily also involves interactions with lipid membrane structures, and thus the creation of DNA-lipid hybrid assemblies. Due to their differing chemical nature, however, highly charged nucleic acids and amphiphilic lipids do not seem the best match for the construction of such systems, and in fact they are rarely found in nature. In recent years, however, a large variety of lipid-interacting DNA conjugates were developed, which are now increasingly being applied also for the realization of DNA nanostructures interacting with lipid bilayer membranes. In this Account, we will present the current state of this emerging class of nanosystems. After a brief overview of the basic biophysical and biochemical properties of lipids and lipid bilayer membranes, we will discuss how DNA molecules can interact with lipid membranes through electrostatic interactions or via covalent modification with hydrophobic moieties. We will then show how such DNA-lipid interactions have been utilized for the realization of DNA nanostructures attached to or embedded within lipid bilayer membranes. Under certain conditions, DNA nanostructures remain mobile on membranes and can dynamically associate into higher order complexes. Hydrophobic modification of DNA nanostructures can further result in intra- or intermolecular aggregation, which can also be utilized as a structural switching mechanism. Appropriate design and chemical modification even allows insertion of DNA nanostructures into lipid bilayer membranes, resulting in artificial ion channel mimics made from DNA. Interactions of DNA nanodevices with living cells also involve interactions with membrane structures. DNA-based nanostructures can be directed to cell surfaces via antibody-antigen interactions, and their cellular uptake can be stimulated by modification with appropriate receptor ligands. In the future, membrane-embedded DNA nanostructures are expected to find application in diverse areas ranging from basic biological research over nanotechnology to synthetic biology.

The fast kinetics of induction and relaxation of bacteriochlorophyll prompt and delayed fluorescence together with absorption changes of the reaction center (RC) dimer (P) were measured by combination of flashes from laser diodes in intact cells of wild type, carotenoidless (R-26) and cytochrome c 2 deficient (CYCA) mutants of photosynthetic bacteria Rhodobacter sphaeroides. The fluorescence induction under high intensity of continuous light splits into fast and slow rises both overlapped by the (carotenoid and/or bacteriochlorophyll) triplet quenching. The fast phase is purely photochemical as it depends strongly on the number of photons absorbed. The slow phase is the combination of thermal and photochemical reactions and reflects the multiple turnover of the system. Upon short flash, the fluorescence yield cannot reach the maximum due to partial reopening of the RCs by rapid donor and acceptor side reactions. Longer flashes are needed to close the RC completely. Contrary to higher plants, the kinetics of induction and relaxation of the fluorescence yield in bacteria are controlled principally by P þ . The reactions on the quinone side play minor role. The quantitative determination of the cyclic electron transfer rate can be based on calibration to the quantity of P þ . 2797-SympDesign and Engineering of a Light-Activated Potassium Channel

The stability of aptamer-ligand complexes is probed in nanopore-based dynamic force spectroscopy experiments. Specifically, the ATP-binding aptamer is investigated using a backward translocation technique, in which the molecules are initially pulled through an α-hemolysin nanopore from the cis to the trans side of a lipid bilayer membrane, allowed to refold and interact with their target, and then translocated back in the trans-cis direction. From these experiments, the distribution of bound and unbound complexes is determined, which in turn allows determination of the dissociation constant Kd ≈ 0.1 mM of the aptamer and of voltage-dependent unfolding rates. The experiments also reveal differences in binding of the aptamer to AMP, ADP, or ATP ligands. Investigation of an aptamer variant with a stabilized ATP-binding site indicates fast conformational switching of the original aptamer before ATP binding. Nanopore force spectroscopy is also used to study binding of the thrombin-binding aptamer to its target. To detect aptamer-target interactions in this case, the stability of the ligand-free aptamer-containing G-quadruplexes-is tuned via the potassium content of the buffer. Although the presence of thrombin was detected, limitations of the method for aptamers with strong secondary structures and complexes with nanomolar Kd were identified.

Nanopore translocation experiments are increasingly applied to probe the secondary structures of RNA and DNA molecules. Here, we report two vital steps toward establishing nanopore translocation as a tool for the systematic and quantitative analysis of polynucleotide folding: 1), Using α-hemolysin pores and a diverse set of different DNA hairpins, we demonstrate that backward nanopore force spectroscopy is particularly well suited for quantitative analysis. In contrast to forward translocation from the vestibule side of the pore, backward translocation times do not appear to be significantly affected by pore-DNA interactions. 2), We develop and verify experimentally a versatile mesoscopic theoretical framework for the quantitative analysis of translocation experiments with structured polynucleotides. The underlying model is based on sequence-dependent free energy landscapes constructed using the known thermodynamic parameters for polynucleotide basepairing. This approach limits the adjustable parameters to a small set of sequence-independent parameters. After parameter calibration, the theoretical model predicts the translocation dynamics of new sequences. These predictions can be leveraged to generate a baseline expectation even for more complicated structures where the assumptions underlying the one-dimensional free energy landscape may no longer be satisfied. Taken together, backward translocation through α-hemolysin pores combined with mesoscopic theoretical modeling is a promising approach for label-free single-molecule analysis of DNA and RNA folding.

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This work shows that short mixmers composed of Locked Nucleic Acid (LNA) and DNA can be used to add functional groups to specific sequences within a double stranded DNA molecule for single molecule manipulation (SMM) experiments. Traditional immobilization protocols often preclude experiments on native biomolecular complexes; the use of these biomolecular LNA handles could broaden the experimental possibilities by providing a simple, nonpertubative labeling strategy. We show triplex-forming LNA probes with functional moities can immobilize DNA stably and specifically with one simple hybridization step. Further investigations of stability show that, although the LNA handles are stable under shear forces up to 10 pN for long times, they are up to two orders of magnitude less stable when force is applied in a geometry which sequentially destabilizes the triplexes (i.e. an unzipping configuration). We provide a set of rules for the esign and use of LNA handles in SMM applications and show results using biomolecular handles on DNA structures from E. coli.