ABSTRACT
We created nanometer-scale transmembrane channels in lipid bilayers by means of self-assembled DNA-based nanostructures. Scaffolded DNA origami was used to create a stem that penetrated and spanned a lipid membrane, as well as a barrel-shaped cap that adhered to the membrane, in part via 26 cholesterol moieties. In single-channel electrophysiological measurements, we found similarities to the response of natural ion channels, such as conductances on the order of 1 nanosiemens and channel gating. More pronounced gating was seen for mutations in which a single DNA strand of the stem protruded into the channel. Single-molecule translocation experiments show that the synthetic channels can be used to discriminate single DNA molecules.
Subject(s)
Cholesterol/chemistry , DNA/chemistry , Ion Channels/chemistry , Lipid Bilayers , Nanostructures , Biosensing Techniques , Electrophysiological Phenomena , Nucleic Acid Conformation , Phosphatidylcholines/chemistryABSTRACT
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.
Subject(s)
DNA/chemistry , DNA/ultrastructure , Models, Chemical , Models, Molecular , Nanostructures/chemistry , Nanostructures/ultrastructure , Base Sequence , Computer Simulation , Molecular Sequence Data , Nucleic Acid Conformation , PorosityABSTRACT
Nanopore force spectroscopy is used to study the unzipping kinetics of two DNA hairpin molecules with a 12 base pair long stem containing two contiguous stretches of six GC and six AT base pairs in interchanged order. Even though the thermodynamic stabilities of the two structures are nearly the same, they differ greatly in their unzipping kinetics. When the GC segment has to be broken before the AT segment, the unfolding rate is orders of magnitude smaller than in the opposite case. We also investigated hairpins with stem regions consisting only of AT or GC base pairs. The pure AT hairpins translocate much faster than the other hairpins, whereas the pure GC hairpins translocate on similar timescales to the hairpins with only an initial GC segment. For each hairpin, nanopore force spectroscopy is performed for different loading rates and the resulting unzipping distributions are mathematically transformed to a master curve that yields the unfolding rate as a function of applied voltage. This is compared with a stochastic model of the unfolding process for the two sequences for different voltages. The results can be rationalized in terms of the different natures of the free energy landscapes for the unfolding process.
Subject(s)
DNA/chemistry , Microscopy, Atomic Force/methods , Models, Chemical , Nanostructures/chemistry , Nanostructures/ultrastructure , Base Sequence , Computer Simulation , Inverted Repeat Sequences , Models, Statistical , Molecular Sequence Data , Nucleic Acid Denaturation , PorositySubject(s)
Emulsions/chemistry , Nanostructures/chemistry , Spectrum Analysis/methods , Electricity , Lipids/chemistry , PorosityABSTRACT
Attempts to construct artificial systems from biological molecules such as DNA and RNA by self-assembly are compatible with the recent development of synthetic biology. Genetic mechanisms can be used to produce or control artificial structures made from DNA and RNA, and these structures can in turn be used as artificial gene regulatory elements, in vitro as well as in vivo. Artificial biochemical circuits can be incorporated into cell-like reaction compartments, which opens up the possibility to operate them permanently out of equilibrium. In small systems, stochastic effects become noticeable and will have to be accounted for in the design of future systems.
