Here, a protocol for the protection of DNA origami nanostructures in Mg-depleted and nuclease-rich media using natural cationic polysaccharide chitosan and synthetic linear polyethyleneimine (LPEI) coatings is presented.
DNA origami nanostructures hold an immense potential to be used for biological and medical applications. However, low-salt conditions and nucleases in physiological fluids induce denaturation and degradation of self-assembled DNA nanostructures. In non-viral gene delivery, enzymatic degradation of DNA is overcome by the encapsulation of the negatively charged DNA in a cationic shell. Herein, inspired by gene delivery advancements, a simple, one-step and robust methodology is presented for the stabilization of DNA origami nanostructures by coating them with chitosan and linear polyethyleneimine. The polycation coating efficiently protects DNA origami nanostructures in Mg-depleted and nuclease-rich media. This method also preserves the full addressability of enzyme- and aptamer-based functionalization of DNA nanostructures.
DNA is a versatile building block for the programmable self-assembly of nanoscale structures1. The most popular method for creating DNA nanostructures is the DNA origami technique, which is based on the self-assembly of a long circular, single stranded DNA scaffold with the aid of hundreds of shorter shape-determining synthetic staple strands2. Today, the creation of DNA nanostructures in almost any geometry and morphology is readily feasible. DNA nanostructures can be site-specifically functionalized with high precision3,4 and can be programmed to undergo allosteric conformational changes5,6. Hence, using DNA as a building material offers the unique opportunity to create programmable and responsive custom-designed nanostructures for applications in biosensing, diagnostics and drug delivery. However, DNA nanostructures are susceptible to digestion by exo- and endo-nucleases, and lattice-based 3D DNA origami nanostructures generally require high salinity buffers (e.g., 5-20 mM Mg+2) to maintain their integrity7,8.
The rapid degradation of DNA by nucleases present in blood and the extracellular matrix is a major obstacle to the efficient in vivo delivery of genetic products into cells9. To overcome this limitation, in non-viral gene delivery, DNA is mixed with a cationic polymer at a defined N/P charge ratio (ratio of amines in polycation to the phosphates in DNA)10. The complex of DNA with a cationic polymer, known as polyplex, protects DNA from nuclease-mediated degradation and enhances its cellular uptake11. Inspired by gene delivery advancements, oligolysine12, oligolysine-polyethylene glycol (PEG) copolymers13, poly (2-dimethylamino-ethylmethacrylate) (PDMAEMA)-based polymers14, and virus capsid proteins15 have been used for stabilizing DNA origami nanostructures.
Recently, we reported a method for the protection of DNA origami nanostructures in Mg-depleted and nuclease-rich media using the natural cationic polysaccharide chitosan and the synthetic linear polyethyleneimine (LPEI) was reported16. This article is an adaptation of our earlier work and describes the detailed protocol for the preparation of polyplexes, their characterization, testing the stability of naked and protected nanostructures in low-salt and nuclease-rich media and examining the addressability of enzyme- and aptamer-functionalized DNA origami nanostructures upon polycation coating.
1. Preparing Polycation Stock Solution
2. Purification of DNA Origami Nanostructures
3. Agarose Gel Electrophoresis (AGE)
4. Negative Stain Transmission Electron Microscopy (nsTEM)
5. Polyplex Formation
NOTE: Illustrative example for preparing a polyplex comprising DNA origami and chitosan at N/P 0.01-8 ratios.
6. Decapsulation with Dextran Sulphate
7. Stability towards Mg Depletion
8. DNase I Titration Assay of Naked DNA Origami Nanostructures
9. Stability of Polyplexes towards DNase I
10. Stability towards Fetal Bovine Serum (FBS)
11. Testing the Addressability of Horseradish Peroxidase Enzyme (HRP)-functionalized DNA Origami upon Coating with Polycations
12. Testing the Addressability of Hemin-binding Aptamers (HBA)-functionalized DNA Origami upon Coating with Polycations
Three DNA origami nanostructures of different configurations were designed, including a nanorod (NR), a nanobottle (NB) and a wireframe nanostructure (WN) (the 3D models of the nanostructures are illustrated in Figure 2). The NR and the NB were designed based on a square and honeycomb lattice, respectively, using caDNAno17 and the WN was created using the Daedalus software18. A comprehensive protocol for the design and self-assembly of DNA nanostructures has been published already19,20,21,22. The shape-specific staple strands were ordered, self-assembled and further purified based on a protocol described by Stahl et al.23 (step 2).
Polyplexes were characterized by the gel retardation assay and nsTEM imaging. Upon mixing the DNA origami with the polycations, a shift in the naked nanostructure band towards the cathode was observed (Figure 1A). This can be attributed to the counterbalancing of the negative charge of phosphate groups upon binding to polycations and the overall size increase of the complex. Adding an extra amount of polyanions such as dextran sulphate can reverse the polyplex formation. In this regard, dextran sulphate of higher molecular weight (MW ~ 40 kDa) showed to be more efficient for the decomplexation compared to lower molecular weight dextran sulphate (MW ~ 4 kDa) (Figure 1B).
While imaging LPEI polyplexes by uranyl acetate negative stain TEM, it was difficult to image any particles. It was hypothesized that LPEI might hinder uranyl acetate from binding to the phosphate backbone due to tight shielding of DNA origami. Therefore, prior to nsTEM imaging, an excess amount of dextran sulphate was added to LEPI polyplexes. The unraveled DNA origami nanostructures showed no sign of defect nor decomposition. In contrary, chitosan polyplexes were successfully imaged with no need for polyanion treatment (Figure 2).
All naked DNA nanostructures (irrespective of their different configurations) were denatured completely after one day of incubation at 37 °C in Mg-zero buffer, as confirmed by nsTEM imaging. In contrast, nanostructures coated with LPEI or chitosan at N/P≥1 remained intact. Similarly, DNase I titration assays showed the susceptibility of unprotected nanostructures towards enzymatic digestion. While naked nanostructures were completely digested in the presence of 1 U/mL DNase I after 2 h, the LEPI or chitosan encapsulated DNA origami stayed intact in Mg-zero buffer supplemented with 10 U/mL DNase I for a minimum of one day (Figure 2). Higher stability towards nucleolytic digestion was achieved by increasing the N/P ratio of the polyplexes. Additionally, LPEI protects the DNA nanostructures more efficiently compared to chitosan (Figure 3A, Figure 3C), probably due to the higher charge density of LEPI, and thus, higher binding affinity towards the DNA24. No difference was observed while using LPEI of different molecular weight (Figure3B).
The addressability of functional groups in DNA nanostructures after encapsulation in a polymer shell is an important feature. Furthermore, the compatibility of polycation coating with the horseradish peroxidase enzyme (HRP) and hemin-binding aptamers (HBA) functionalized DNA origami was examined. Three biotinylated staple strands were protruded from the surface of the NR and then functionalized with HRP conjugated streptavidin (three enzymes per DNA origami). The catalytically highly active (Kd: 439 nM) HBA aptamer PS2.M (5'-GTGGGTAGGGCGGGTGG-3') was chosen for these experiments25. Up to 24 staple strands were protruded from the NR surface with a 5-nm length linker (5'-AAAAGAAAAGAAAAA-3') followed by the PS2.M sequence (24 aptamers per DNA origami). No remarkable hindrance of enzymatic or aptamer activity was observed upon coating HRP- or HBA-functionalized DNA origami with LPEI and chitosan at variant N/P ratios (Figure 4A-B)16. However, the kinetic of HRP enzyme has been dramatically changed after binding to the DNA origami (Figure 4C).
Figure 1: Representative AGE images of polyplex formation and decapsulation. (A) The electrophoretic mobility shift assay for NB mixed with chitosan at N/P ratios of 0.01-8. Each lane contains 1 nmol NB. The first lane is the reference NB. (B) Decapsulation of polyplexes by polyanionic dextran sulphate (DS). This figure has been modified from a previously published figure16. Please click here to view a larger version of this figure.
Figure 2: Negative stain TEM micrographs of DNA origami and polyplexes. 3D model and nsTEM micrographs of naked DNA origami nanostructures (columns 1 and 2). nsTEM micrographs of LPEI polyplexes (after decapsulation) and chitosan polyplexes (columns 3 and 4). nsTEM images of naked and protected DNA origami (polyplex) subjected to Mg depletion (columns 5, 6 and 7), enzymatic degradation (columns 8 and 9), serum digestion (columns 10 and 11) for one day at 37 °C. LPEI-5 kDa was used in this assay. Naked DNA nanostructures were degraded beyond detection on an AGE in the presence of 1 U/mL DNase I or in TB + 10% FBS after 2 h of incubation at 37 °C. Scale bars: 100 nm. This figure has been modified from a previously published figure16. Please click here to view a larger version of this figure.
Figure 3: Representative results of DNase I protection assays. (A) The DNase I protection assay for polyplexes of WN with LPEI-5 kDa, LPEI-10 kDa and LPEI-25 kDa prepared at N/P ratio of 2, 4, 8 and 10. The samples were subjected to 10 U/mL DNase I for 24 h at 37 °C. The last lane is the control WN. The polyplexes were decapsulated prior to loading into the gel. (B) The normalized mean band intensity for polyplexes of DNA origamis with different LPEI extracted from AGE image-A. Y axis represents normalized mean band intensity. (C) The DNase I protection assay of chitosan-WN polyplexes prepared at N/P ratios of 1, 2, 4, 10 and 20. The samples were subjected to 10 U/mL DNase I for 24 h at 37 °C. Lane 6 is the control polyplex (N/P 20). The last lane is the control WN. All chitosan polyplexes were decapsulated prior loading into the gel. This figure has been modified from a previously published figure16. Please click here to view a larger version of this figure.
Figure 4:Â Representative results of colorimetric enzyme- and aptamer-functionalized DNA origami assays. (A) The colorimetric assay of enzyme-functionalized nanostructures (HRP-NR) coated with chitosan 3.7 h after the reaction start. (B) Colorimetric assay of aptamer-functionalized nanostructures (HBA-NR) coated with chitosan, 6 min after the reaction initiation. Y axis represents the normalized absorbance. (C) Monitoring the colorimetric assay of HRP and HRP-NR over time. This figure has been modified from a previously published figure16. Please click here to view a larger version of this figure.
Cationic polymers | Chitosan | LPEI-5 kDa | LPEI-10 kDa | LPEI-25 kDa |
Molecular weight of polymer (kDa) | 5 | 5 | 10 | 25 |
Molecualr weight of monomer (g/mol) | 161 | 43 | 43 | 43 |
Number of amine per monomer | 1 | 1 | 1 | 1 |
Number of amine per polymer | 28 | 116 | 232 | 581 |
Table 1. Calculating the number of amine groups per polycation.
Molecular weight of dextran sulphate (kDa) | 4 | 40 |
Molecular weight of monomer (g/mol) | 366 | 366 |
Number of sulphate per monomer | 2 | 2 |
Number of sulphate per polymer | 22 | 220 |
Table 2. Calculating the number of sulphate groups per polyanion.
In the self-assembly of DNA origami, the staple strands are typically added in 5-10 excess ratio to the scaffold. These excess staple strands also bind to the polycation and form polyplexes in the monometer range which are further difficult to be separated from DNA origami polyplexes. Hence, a critical step in polyplex formation is to remove the excess staple strands and use well-purified DNA origami nanostructures.
Other methods have been developed for stabilizing DNA nanostructures such as the cyclization of DNA strands via a click reaction26. As alkyne- and azide-modified staple strands are required for the formation of interlocked single-stranded rings, this technique is limited for the stabilization of small nanostructures such a DNA catenane, and it is not readily scalable for larger DNA origami structure such as those used in this protocol. Recently, Gerling et al.27 reported a method for creating covalent cyclobutene pyrimidine dimer (CPD) bonds between neighboring thymidines within DNA nanostructures using ultraviolet irradiation. Although this method is site-selective and scalable, its efficiency in protecting DNA origami is much lower than polycation coating. For example, CPD-stabilized DNA origami objects endure only 0.4 U/mL DNase I for 1 h, while polyplexes were stable in the presence of 10 U/mL DNase I for at least one day.
In brief, gene-therapy inspired chitosan and LPEI coating is a low-cost, one-step and efficient method to address the long-term stability of DNA origami nanostructures in Mg-depleted and nuclease-rich media. The reversibility of the polyplex formation facilitates its application in multi-step diagnostic tests where the protection of DNA origami against nucleolytic degradation or salt-depletion is crucial in one step but may cause problems in other steps such as DNA amplification. Additionally, polycation coating is a potential approach for enhancing the cellular uptake of DNA origami nanostructures for drug-delivery applications28.
This project has received funding from the European Union's Horizon 2020 research and innovation program under grant agreement No. 686647. TEM images were recorded on a Morgagni operated at 80 kV at the EM Facility of the Vienna Biocenter Core Facilities GmbH (VBCF). We would like to thank Tadija Kekic for assistance in the graphical design and Elisa De LIano for designing the wireframe nanostructure.
Name | Company | Catalog Number | Comments |
10× DNase I reaction buffer | New England Biolab (NEB) | B0303 | |
ABTS (2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) | Alfa Aesar | J65535 | |
Amicon ultracentrifugation columns | Merck Millipore | UCF500308, UCF510024 | |
Chitosan oligosaccharide lactate (Mn ~ 4000-6000, > 90% deacetylated, 60%. composition oligosaccharide | Sigma-Aldrich | 523682 | |
Design-specific staple strands | Integrate DNA Technologies (IDT) | ||
Dextran sulfate sodium salt (Mr ~ 4 kDa) | Sigma-Aldrich | 75027 | |
Dextran sulfate sodium salt (Mr ~ 40 kDa) | Sigma-Aldrich | 42867 | |
DNA gel loading dye (6×) | ThermoFischer sceintific | R0611 | |
DNase I (RNase free) | New England Biolab (NEB) | M0303 | |
Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) | Sigma-Aldrich | E3889 | |
Ethylenediaminetetraacetic acid (EDTA) BioUltra, anhydrous, ≥99% (titration) | Sigma-Aldrich | EDS | |
Freeze'N Squeeze DNA Gel Extraction Spin Columns | Biorad | 7326165 | |
Hemin | Sigma-Aldrich | H9039 | |
HEPES | Sigma-Aldrich | H3375 | |
Hydrogen peroixde slution (32 wt%) | Sigma-Aldrich | 216763 | |
LPEI-25 kDa | Polysciences, Inc | 23966-1 | |
NuPAGE Sample Reducing Agent (500 mM dithiothreitol (DTT)) | ThermoFischer sceintific | NP0004 | |
Polyethylene glycol (PEG, MW ~ 8000 Da) | Carl Roth | O263.1 | |
Polyethylenimine, linear, average Mn 10,000, PDI ≤1.2 | Sigma-Aldrich | 765090 | |
Polyethylenimine, linear, average Mn 5,000, PDI ≤1.3 | Sigma-Aldrich | 764582 | |
Proteinase K solution (20 mg/ml) | ThermoFischer sceintific | AM2548 | |
Streptavidin-conjugated HRP | ThermoFischer sceintific | N100 | |
SYBER safe DNA gel stain | ThermoFischer sceintific | S33102 |
This article has been published
Video Coming Soon
ABOUT JoVE
Copyright © 2024 MyJoVE Corporation. All rights reserved