Purification of DNA-origami nanostructures by rate-zonal centrifugation.
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ABSTRACT:
SUBMITTER: Lin C
PROVIDER: S-EPMC3553994 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:DNA origami purification is essential for many fields, including biophysics, molecular engineering, and therapeutics. The increasing interest in DNA origami has led to the development of rate-zonal centrifugation (RZC) as a scalable, high yield, and contamination-free method for purifying DNA origami nanostructures. RZC purification uses a linear density gradient of viscous media, such as glycerol or sucrose, to separate molecules according to their mass and shape. However, many methods for creating density gradients are time-consuming because they rely on slow passive diffusion. To expedite the preparation time, we used a LEGO gradient mixer to generate rotational motion and rapidly create a quasi-continuous density gradient with a minimal layering of the viscous media. Rotating two layers of differing concentrations at an angle decreases the time needed to form the density gradient from a few hours to minutes. In this study, the density gradients created by the LEGO gradient mixer were used to purify 3 DNA origami shapes that have different aspect ratios and numbers of components, with an aspect ratio ranging from 1:1 to 1:100 and the number of components up to 2. The gradient created by our LEGO gradient mixer is sufficient to purify folded DNA origami nanostructures from excess staple strands, regardless of their aspect ratios. Moreover, the gradient was able to separate DNA origami dimers from DNA origami monomers. In light of recent advances in large-scale DNA origami production, our method provides an alternative for purifying DNA origami nanostructures in large (gram) quantities in resource-limited settings.
| S-EPMC10355445 | biostudies-literature
Project description:BackgroundCentrifugation is commonly used as a first step to enrich biomarkers from blood. Biomarkers are separated on the basis of density and/or diameter. However, the centrifugation protocol affects the yield and purity of biomarkers, for example, isolation of platelets results in co-isolation with extracellular vesicles (EVs).ObjectiveTo assess the ability of rate zonal centrifugation (RZC) to separate platelets from co-isolated EVs.MethodsUsing a linear Optiprep gradient, RZC was able to separate a mixture of beads with different diameters but similar density. Next, RZC was applied to samples containing both platelets and platelet-derived EVs (n = 3). After RZC, all fractions were collected and stained with anti-CD61-Alexa 488 to measure the concentrations of platelets and platelet-derived EVs by flow cytometry.ResultsWe confirm that RZC separates polystyrene beads with diameters of 140 nm, 380 nm and 1,000 nm. Next, we show that the majority of platelets occur in fractions 8-19, whereas the majority of platelet-derived EVs are detectable in fractions 1-7. Furthermore, each fraction contains a different diameter range of platelets, which suggests that separation is indeed diameter based.ConclusionRZC can partially separate platelets from EVs.
| S-EPMC7443435 | biostudies-literature
Project description:DNA origami is a powerful tool to fold 3-dimensional DNA structures with nanometer precision. Its usage, however, is limited as high ionic strength, temperatures below ∼60 °C, and pH values between 5 and 10 are required to ensure the structural integrity of DNA origami nanostructures. Here, we demonstrate a simple and effective method to stabilize DNA origami nanostructures against harsh buffer conditions using [PdCl4]2-. It provided the stabilization of different DNA origami nanostructures against mechanical compression, temperatures up to 100 °C, double-distilled water, and pH values between 4 and 12. Additionally, DNA origami superstructures and bound cargos are stabilized with yields of up to 98%. To demonstrate the general applicability of our approach, we employed our protocol with a Pd metallization procedure at elevated temperatures. In the future, we think that our method opens up new possibilities for applications of DNA origami nanostructures beyond their usual reaction conditions.
| S-EPMC10905993 | biostudies-literature
Project description:An orthogonal, noncovalent approach to direct the assembly of higher-order DNA origami nanostructures is described. By incorporating perfluorinated tags into the edges of DNA origami tiles we control their hierarchical assembly via fluorous-directed recognition. When we combine this approach with Watson-Crick base-pairing we form discrete dimeric constructs in significantly higher yield (8x) than when either molecular recognition method is used in isolation. This integrated "catch-and-latch" approach, which combines the strength and mobility of the fluorous effect with the specificity of base-pairing, provides an additional toolset for DNA nanotechnology, one that enables increased assembly efficiency while requiring significantly fewer DNA sequences. As a result, our integration of fluorous-directed assembly into origami systems represents a cheap, atom-efficient means to produce discrete superstructures.
| S-EPMC9835977 | biostudies-literature
Project description:DNA origami has emerged in recent years as a powerful technique for designing and building 2D and 3D nanostructures. While the breadth of structures that have been produced is impressive, one of the remaining challenges, especially for DNA origami structures that are intended to carry out useful biomedical tasks in vivo, is to endow them with the ability to detect and respond to molecules of interest. Target molecules may be disease indicators or cell surface receptors, and the responses may include conformational changes leading to the release of therapeutically relevant cargo. Nucleic acid aptamers are ideally suited to this task and are beginning to be used in DNA origami designs. In this review, we consider examples of uses of DNA aptamers in DNA origami structures and summarise what is currently understood regarding aptamer-origami integration. We review three major roles for aptamers in such applications: protein immobilisation, triggering of structural transformation, and cell targeting. Finally, we consider future perspectives for DNA aptamer integration with DNA origami.
| S-EPMC6315403 | biostudies-other
Project description:The aggregation of human islet amyloid polypeptide (hIAPP) plays a major role in the pathogenesis of type 2 diabetes mellitus (T2DM), and numerous strategies for controlling hIAPP aggregation have been investigated so far. In particular, several organic and inorganic nanoparticles (NPs) have shown the potential to influence the aggregation of hIAPP and other amyloidogenic proteins and peptides. In addition to conventional NPs, DNA nanostructures are receiving more and more attention from the biomedical field. Therefore, in this work, we investigated the effects of two different DNA origami nanostructures on hIAPP aggregation. To this end, we employed in situ turbidity measurements and ex situ atomic force microscopy (AFM). The turbidity measurements revealed a retarding effect of the DNA nanostructures on hIAPP aggregation, while the AFM results showed the co-aggregation of hIAPP with the DNA origami nanostructures into hybrid peptide-DNA aggregates. We assume that this was caused by strong electrostatic interactions between the negatively charged DNA origami nanostructures and the positively charged peptide. Most intriguingly, the influence of the DNA origami nanostructures on hIAPP aggregation differed from that of genomic double-stranded DNA (dsDNA) and appeared to depend on DNA origami superstructure. DNA origami nanostructures may thus represent a novel route for modulating amyloid aggregation in vivo.
| S-EPMC7694230 | biostudies-literature
Project description:1. Rat liver mitochondria were separated on the basis of their sedimentation coefficients in an iso-osmotic gradient of Ficoll-sucrose by rate zonal centrifugation. The fractions (33, each of 40ml) were collected in order of decreasing density. Fractions were analysed by spectral analysis to determine any differences in the concentrations of the cytochromes and by enzyme analyses to ascertain any differences in the activities of NADH dehydrogenase, succinate dehydrogenase and alpha-glycerophosphate dehydrogenase. 2. When plotted as% of the highest specific concentration, the contents of cytochrome a+a(3) and cytochrome c+c(1) were constant in all fractions but cytochrome b was only 65% of its maximal concentration in fraction 7 and increased with subsequent fractions. As a result, the cytochrome b/cytochrome a+a(3) ratio almost doubled between fractions 7 and 25 whereas the cytochrome c+c(1)/cytochrome a+a(3) ratio was unchanged. 3. Expression of the dehydrogenase activities as% of highest specific activity showed the following for fractions 6-26: NADH dehydrogenase activity remained fairly constant in all fractions; succinate dehydrogenase activity was 62% in fraction 6 and increased steadily to its maximum in fraction 18 and then decreased; the activity of alpha-glycerophosphate dehydrogenase was only 53% in fraction 6 and increased slowly to its peak in fractions 22 and 24. 4. These differences did not result from damaged or fragmented mitochondria or from microsomal contamination. 5. These results demonstrate that isolated liver mitochondria are biochemically heterogeneous. The importance of using a system for separating biochemically different mitochondria in studies of mitochondrial biogenesis is discussed.
| S-EPMC1174058 | biostudies-other
Project description:Insulin binds the insulin receptor (IR) and regulates anabolic processes in target tissues. Impaired IR signalling is associated with multiple diseases, including diabetes, cancer and neurodegenerative disorders. IRs have been reported to form nanoclusters at the cell membrane in several cell types, even in the absence of insulin binding. Here we exploit the nanoscale spatial organization of the IR to achieve controlled multivalent receptor activation. To control insulin nanoscale spatial organization and valency, we developed rod-like insulin-DNA origami nanostructures carrying different numbers of insulin molecules with defined spacings. Increasing the insulin valency per nanostructure markedly extended the residence time of insulin-DNA origami nanostructures at the receptors. Both insulin valency and spacing affected the levels of IR activation in adipocytes. Moreover, the multivalent insulin design associated with the highest levels of IR activation also induced insulin-mediated transcriptional responses more effectively than the corresponding monovalent insulin nanostructures. In an in vivo zebrafish model of diabetes, treatment with multivalent-but not monovalent-insulin nanostructures elicited a reduction in glucose levels. Our results show that the control of insulin multivalency and spatial organization with nanoscale precision modulates the IR responses, independent of the insulin concentration. Therefore, we propose insulin nanoscale organization as a design parameter in developing new insulin therapies.
| S-EPMC10873203 | biostudies-literature
Project description:DNA origami technology enables the folding of DNA strands into complex nanoscale shapes whose properties and interactions with molecular species often deviate significantly from that of genomic DNA. Here, we investigate the salting-out of different DNA origami shapes by the kosmotropic salt ammonium sulfate that is routinely employed in protein precipitation. We find that centrifugation in the presence of 3 M ammonium sulfate results in notable precipitation of DNA origami nanostructures but not of double-stranded genomic DNA. The precipitated DNA origami nanostructures can be resuspended in ammonium sulfate-free buffer without apparent formation of aggregates or loss of structural integrity. Even though quasi-1D six-helix bundle DNA origami are slightly less susceptible toward salting-out than more compact DNA origami triangles and 24-helix bundles, precipitation and recovery yields appear to be mostly independent of DNA origami shape and superstructure. Exploiting the specificity of ammonium sulfate salting-out for DNA origami nanostructures, we further apply this method to separate DNA origami triangles from genomic DNA fragments in a complex mixture. Our results thus demonstrate the possibility of concentrating and purifying DNA origami nanostructures by ammonium sulfate-induced salting-out.
| S-EPMC8911265 | biostudies-literature
Project description:Density gradient centrifugation is an effective method for the isolation and purification of small particles. Hollow rotors capable of hosting density gradients replace the need for centrifuge tubes and therefore allow separations at large scales. So far, zonal rotors have been used for biological separations ranging from the purification of whole cells down to serum proteins. We demonstrate that the high-resolution separation method opens up exciting perspectives apart from biology, namely in sorting mixtures of synthetic nanoparticles. Loading and unloading, while the rotor is spinning, avoids perturbations during acceleration and deceleration periods, and thus makes a vital contribution to sorting accuracy. Nowadays one can synthesize nanoscale particles in a wide variety of compositions and shapes. A prominent example for this are "colloidal molecules" or, generally speaking, defined assemblies of nanoparticles that can appear in varying aggregation numbers. Fractionation of such multimodal colloids plays an essential role with regard to their organization into hierarchical organized superstructures such as films, mesocrystals and metamaterials. Zonal rotor centrifugation was found to be a scalable method of getting "colloidal molecules" properly sorted. It allows access to pure fractions of particle monomers, dimers, and trimers, just as well as to fractions that are essentially rich in particle tetramers. Separations were evaluated by differential centrifugal sedimentation, which provides high-resolution size distributions of the collected nanoparticle fractions. The performance achieved in relation to resolution, zone widths, sorting efficiencies and recovery rates clearly demonstrate that zonal rotor centrifugation provides an excellent solution to the fractionation of nanoparticle mixtures.
| S-EPMC9070787 | biostudies-literature