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.
Display omitted
•ssDNA scaffolds are purified by anion-exchange or multimodal chromatography.•2-step NaCl elution is used to isolate ssDNA from asymmetric PCR impurities.•With anion-exchange ...chromatography ssDNA is eluted on the first step (64 mS/cm)•With multimodal chromatography ssDNA is eluted on the second step (147 mS/cm)•Recovered ssDNA scaffolds can be used to assemble DNA-origami nanostructures.
DNA-origami biomanufacturing relies in many cases on the use of asymmetric PCR (aPCR) to generate 500–3500 base, object-specific, single-stranded DNA (ssDNA) scaffolds. Each scaffold is usually purified by agarose gel extraction, a technique that is laborious, limited, not scalable, presents low recovery yields and a low-quality product. Alternatively, we present a chromatography-based method to purify ssDNA scaffolds from aPCR mixtures, which can be used in the context of DNA-origami techniques.
aPCR was performed to generate single and double-stranded DNA (dsDNA) from the M13mp18 genome. To isolate the target ssDNA from dsDNA and other PCR impurities, anion-exchange (Q-ligand) and multimodal chromatography (CaptoTM adhere ImpRes) were explored using stepwise gradients with increasing NaCl concentrations. In anion exchange chromatography, the less-charged ssDNA eluted before the dsDNA. In multimodal chromatography, however, the elution pattern was reversed, highlighting the importance played by hydrophobicity. In either case, collected ssDNA-containing fractions were homogeneous and impurity free.
Finally, 8.4 μg of a 1000-nt ssDNA fragment was purified and used alongside with site-specific short oligonucleotides (staples) to assemble 63-bp edge length tetrahedrons. Gel electrophoresis showed high assembly yield and purity, whereas fluorescence correlation spectroscopy confirmed that the tetrahedrons had a diffusion coefficient (26.7 μm2 s−1) consistent with the expected size (20 nm).
The chiral state of a molecule plays a crucial role in molecular recognition and biochemical reactions. Because of this and owing to the fact that most modern drugs are chiral, the sensitive and ...reliable detection of the chirality of molecules is of great interest to drug development. The majority of naturally occurring biomolecules exhibit circular dichroism (CD) in the UV range. Theoretical studies and several experiments have demonstrated that this UV-CD can be transferred into the plasmonic frequency domain when metal surfaces and chiral biomolecules are in close proximity. Here, we demonstrate that the CD transfer effect can be drastically enhanced by placing chiral molecules, here double-stranded DNA, inside a plasmonic hotspot. By using different particle types (gold, silver, spheres, and rods) and by exploiting the versatility of DNA origami, we were able to systematically study the impact of varying particle distances on the CD transfer efficiency and to demonstrate CD transfer over the whole optical spectrum down to the near-infrared. For this purpose, nanorods were also placed upright on DNA origami sheets, forming strong optical antennas. Theoretical models, demonstrating the intricate relationships between molecular chirality and achiral electric fields, support our experimental findings. From both experimental measurements and theoretical considerations, we conclude that the transferred CD is most intensive for systems with strong plasmonic hotspots, as we find them in relatively small gaps (5–12 nm) between spherical nanoparticles and preferably between the tips of nanorods.
Optical nanoantennas are known to focus freely propagating light and reversely to mediate the emission of a light source located at the nanoantenna hotspot. These effects were previously exploited ...for fluorescence enhancement and single-molecule detection at elevated concentrations. We present a new generation of self-assembled DNA origami based optical nanoantennas with improved robustness, reduced interparticle distance, and optimized quantum-yield improvement to achieve more than 5000-fold fluorescence enhancement and single-molecule detection at 25 μM background fluorophore concentration. Besides outperforming lithographic optical antennas, DNA origami nanoantennas are additionally capable of incorporating single emitters or biomolecular assays at the antenna hotspot.
Detecting small sequences of RNA in biological samples such as microRNA or viral RNA demands highly sensitive and specific methods. Here, a reconfigurable DNA origami template has been used where a ...chiral arrangement of gold nanorods on the structure can lead to the generation of strong circular dichroism (CD). Switching of the cross‐like DNA structure is achieved by the addition of nucleic acid sequences, which arrests the structure in one of the possible chiral states by specific molecular recognition. A specific sequence can thus be detected through the resulting changes in the plasmonic CD spectrum. We show the sensitive and selective detection of a target RNA sequence from the hepatitis C virus genome. The RNA binds to a complementary sequence that is part of the lock mechanism, which leads to the formation of a defined state of the plasmonic system with a distinct optical response. With this approach, we were able to detect this specific RNA sequence at concentrations as low as 100 pm.
A gold–DNA origami template can show strong circular dichroism (CD). Switching of the cross‐like DNA structure is achieved by the addition of nucleic acid sequences, which arrests the structure in one of the possible chiral states by specific molecular recognition. Specific RNA sequences at concentrations as low as 100 pm can be detected through the resulting changes in the plasmonic CD spectrum.
Delivery of proteins to carry out desired biological functions is a direct approach for disease treatment. However, protein therapy is still facing challenges due to low delivery efficiency, poor ...targeting during trafficking, insufficient therapeutic efficacy, and possible toxicity induced by carriers. Here, we present a novel delivery platform based on DNA origami nanostructure that enables tumor cell transportation of active proteins for cancer therapy. In our design, cytotoxic protein ribonuclease (RNase) A molecules are organized on the rectangular DNA origami nanosheets, which work as nanovehicles to deliver RNase A molecules into the cytoplasm and execute their cell-killing function inside the tumor cells. Cancer cell-targeting aptamers are also integrated onto the DNA origami-based nanoplatform to enhance its targeting effect. This DNA origami-protein coassembling strategy can be further developed to transport other functional proteins and therapeutic components simultaneously for synergistic effects and be adapted for integrated diagnostics and therapeutics.
Application of DNA origami in nanobiomedicine Wang, J; Zhang, P; Xia, Q ...
Nan fang yi ke da xue xue bao = Journal of Southern Medical University,
06/2021, Letnik:
41, Številka:
6
Journal Article
DNA origami technology allows for the precise nanoscale assembly of chemical entities that give rise to sophisticated functional materials. We have created a versatile DNA origami nanofork antenna ...(DONA) by assembling Au or Ag nanoparticle dimers with different gap sizes down to 1.17 nm, enabling signal enhancements in surface-enhanced Raman scattering (SERS) of up to 1011. This allows for single-molecule SERS measurements, which can even be performed with larger gap sizes to accommodate differently sized molecules, at various excitation wavelengths. A general scheme is presented to place single analyte molecules into the SERS hot spots using the DNA origami structure exploiting covalent and noncovalent coupling schemes. By using Au and Ag dimers, single-molecule SERS measurements of three dyes and cytochrome c and horseradish peroxidase proteins are demonstrated even under nonresonant excitation conditions, thus providing long photostability during time-series measurement and enabling optical monitoring of single molecules.
Dextran‐rich droplets are generated in a polyethylene glycol‐rich phase via liquid‐liquid phase separation. This study demonstrates that the dextran‐rich droplets can selectively uptake DNA ...nano/microstructures based on size and shape. This property is explored for constructing a versatile method for purifying DNA nano/microstructures. This method can be applied to various DNA nano/microstructures, including hexagons, triangles, and fibers, with a high yield and short preparation time. This picture shows the time variation of a spherical dextran‐rich droplet capturing DNA nano/microstructures. More information can be found in the Research Article by M. Takinoue et al.
While understanding translocation of DNA through a solid-state nanopore is vital for exploiting its potential for sensing and sequencing at the single-molecule level, surprisingly little is known ...about the dynamics of the propagation of DNA through the nanopore. Here we use linear double-stranded DNA molecules, assembled by the DNA origami technique, with markers at known positions in order to determine for the first time the local velocity of different segments along the length of the molecule. We observe large intramolecular velocity fluctuations, likely related to changes in the drag force as the DNA blob unfolds. Furthermore, we observe an increase in the local translocation velocity toward the end of the translocation process, consistent with a speeding up due to unfolding of the last part of the DNA blob. We use the velocity profile to estimate the uncertainty in determining the position of a feature along the DNA given its temporal location and demonstrate the error introduced by assuming a constant translocation velocity.
PDF
