Fabrication of DNA nanotubes using origami-based nanostructures with sticky ends
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Described here is a simplified method for fabrication of DNA nanotubes using a minimum numbers of staple oligomers for DNA origami. For this purpose, the cylindrical nanotemplates with two sticky ends have been designed using caDNAno software. Then, the nanostructures were shaped in an optimized experimental condition via an origami-based self-assembly reaction. Finally, the produced nanostructures were joined together through their sticky ends using a ligase enzyme. Transmission electron microscope confirmed fabrication of these elongated nanotubes. In addition, high-resolution microscopy of DNA nanotubes by scanning tunnelling microscope indicated efficient attachments of the primarily DNA nanostructures via their sticky ends. The results demonstrated that a ligase treatment of cylindrical DNA nanostructures with the sticky ends made DNA nanotubes with standard shapes using minimum numbers of staples.
KeywordsDNA nanotube T4 ligase Sticky ends TEM STM
DNA nanotechnology uses DNA as a building block to create nano-scale structures which acts as a candidate in multidisciplinary applications such as fabrications of nanodevices, nanoarrays, nanodiagnostics, nanotherapeutics, and the other demanding areas [1, 2, 3, 4]. Among DNA nanostructures, DNA nanotubes have particularly potentials for functions due to high aspect ratio , controllability in fabrications [6, 7], and some capabilities for applications as nanocarriers , nanoarrays , nanowires , nanotools for structural analysis , and nanoanalytes for controlling some fabrication processes .
DNA origami was introduced as a new growth mechanism for DNA assembly for fabrication of various shapes of DNA nanostructures. In this technique, a long single-stranded DNA templated the final structure, so-called DNA scaffold . DNA origami gives excellent control over the size and shape of objects [14, 15]. However, this level of structural complexity requires hundreds of staple strands with different sequence as well as long annealing times . In other words, obtaining nanotubes with high aspect ratio need more quantities of staple oligonucleotides within a DNA origami procedure; however, more staples in fabrication made more costs in practice. Hence, we aimed here to introduce a simple procedure for fabrication of DNA nanotubes via an origami-based technique assisted with a ligase reaction. This fabrication process provides DNA nanotube with minimum numbers of the staples within a self-assembly reaction.
The cylindrical nanostructures were constructed using M13mp18 DNA as scaffold and staples with specific sequences. The nanotemplates jointed together to form DNA nanotubes via a ligase reaction.
Materials and methods
Chemicals and instruments
Thermal condition for self-assembly in origami reaction was set using thermocycler CA 1000 (Bio-Rad, USA). Gel electrophoresis experiments were performed using electrophoresis mini set from Bio-Rad (USA). The micrographs were obtained by NAMA-STM SS-3 (Nano System Pars Corporation, Natsyco, Iran) and TEM (Philips EM028, USA). Highly ordered pyrolytic graphite (HOPG) was prepared from Nano System Pars Corporation. M13mp18 phage genome was purchased from New England Biolabs (USA). T4 DNA ligase was provided by Thermo (USA) and the desired single strand oligonucleotides were synthesised by Sigma. SYBRGold gel staining solution was purchased from Molecular Probes Inc. (Oregon, USA). Quantum Prep™ Freeze ‘N Squeeze DNA Gel Extraction Spin Columns were from Bio-Rad, USA.
Computational modelling of nanotube-based templates with sticky ends
Recently caDNAno is an attractive software for scientists to design variable origami shapes. It commonly applies two inputs, including a lengthy scaffold and a group of crossing staples. M13mp18 DNA was used as DNA scaffold and staples were designed based on their complementarities with special sites of the scaffold sequences for shaping sticky-ended nanostructures.
Self-assembly of nanotube-based templates
Assembly of DNA origami nanotubes was accomplished by mixing 10 nM M13mp18 DNA with 100 nM of each staple oligonucleotide. This self-assembly mixture contained TAE–Mg2+ (40 mM Tris, 20 mM acetic acid, 2 mM EDTA and 12.5 mM magnesium acetate, pH 8.0). Thermal annealing program was applied via a hybridization program from 90 to 25 °C with 1 °C/min ramping rate.
Ligase treatment of the nanotube templates
Ligation was the next step of the fabrication method that resulted longitudinal binding of primarily short DNA nanotubes together. For this purpose, 10 µl origami product, 2 µl 10× T4 DNA ligation buffer, 2 µl 50 % PEG 4,000 solution, 5 U T4 DNA ligase, and nuclease-free water were mixed up to 20 µl in a total volume. The mixture was incubated for 1 h at 22 °C according to the manufacturer’s instruction.
The origami product was loaded into the gel electrophoresis wells after completing the folding phase immediately. The experiment was performed in 1 % agarose gel at TAE buffer with a voltage of 100 V applied in 25 min by power supply. Gel staining was performed by SYBRGold working solution. In addition, the ligation product was run in the same experimental condition for the gel electrophoresis. After electrophoresis, DNA nanotubes were extracted from the gels using Quantum Prep™ Freeze ‘N Squeeze DNA Gel Extraction Spin Columns according to the manual instruction.
Transmission electron microscopy of DNA nanotubes
Transmission electron microscope was used to determine the size and morphology of DNA nanotubes. For this purpose, the samples were immobilized by syringe spraying on agar scientific holey carbon film with 300 mesh Cu(50).
Characterization of DNA nanotubes by STM
The ligation product-contained DNA nanotubes were diluted 103 folds in TAE–Mg2+ buffer (pH 8). Then, 5 μl diluted sample was immobilized on the HOPG by drying for 3 h at room temperature . The samples were imaged using topographic mode with STM, with 0.1 nA current set point and 0.2 V sample bias through a platinum iridium tip. Rough data were first processed using line adjust, plain adjust and average filters of the NAMA-STM Nanoanalyzer software. Then, the colouring process was tested on the obtained micrographs for different levels .
DNA nanotube templates with sticky ends
Staple oligomers for self-assembly of DNA nanotemplates
The 24 staples were indicated with different colours in the computational model. The computerized nanotube contained two sticky ends that those could be jointed to their complements for shaping DNA nanotubes via a ligase reaction.
Electrophoresis behaviours of DNA nanotubes
STM micrographs of DNA nanotubes
DNA as nanomaterial has flexibility to make programmable shapes resulting via base-pairing rules in DNA assembly phenomenon [20, 21, 22]. There are two ways to form DNA nanostructures; first by making a large structure from specialized oligonucleotides known as a bottom-up approach in fabrication ; second by shaping a large molecule that will create a programmable structure that needs to be minimized as a top-down approach in fabrication . According to the ways in DNA nanofabrication, various shapes of nano-scaled DNA structures have been created in octahedron , cubic , icosahedron , and tube [28, 29, 30, 31] forms.
Nanotubes are promising nanomaterials not only at the nano-scale applications, but also they can play a functional role at visual scales, according to their components and designs; however, all of these applications are taking advantages of nanotubes properties, such as their high aspect ratio and size variation [1, 2, 3, 4]. A beneficial material creating these nanostructures may be DNA, with the highest specificity in the assembly and the most simplicity in their fabrications and organizations. Moreover, various softwares have been employed for modelling different structures of DNAs that those would be easier for use in DNA nanotubes [24, 31].
In practice, thermal self-assembly and the isothermal assembly using denaturing agents  were two ways for creating DNA nanostructures; however, many staple oligonucleotides would be needed in the self-assembly of the nanotubes and lead to more costs for obtaining the nanotubes with more lengths. On the other hand, the obtained products would be labile particularly for imaging by electron or scanning probe microscopies [28, 33].
Here, we introduced a fabrication way accomplished with the origami technique and ligation for providing DNA nanotubes with standard dimensions. The diameter of DNA nanotubes could control according to the numbers of circles selected in caDNAno software. In this study, six graphical circles were selected for shaping cylindrical nanotemplates of DNA nanotubes. Moreover, it seems more length of DNA nanotube shaped via selecting more lengths of cylindrical nanotemplates designed by caDNAno software. This procedure made beneficial nanofabrication of nanotubes by employing minimum staples. In addition, this procedure for fabrication of DNA nanotubes would provide a suitable condition for a high-resolution imaging by STM; however, to our knowledge, STM characterization of DNA nanotubes has not been reported, previously.
The nanotubes were resulted via periodic ligation of the primarily self-assembled nanotemplates; however, the gel electrophoresis experiments also confirmed the different weights between the primarily templates of DNA nanotubes and the ligated forms.
Ligation could make elongation of the primarily nanotubes via connecting the sticky ends and their growth in length; hence, T4 ligase could also stabilized DNA nanotubes; make them more robust for imaging by STM. Alternatively, similar report on the tile-based lattices and their ligation for more fully ligatable DNA lattices has been published, previously ; however, the construction of DNA nanotubes in that report has not been shaped directly via caDNAno-based origami self-assembly described here.
Because of the high aspect ratio of DNA nanotubes, these nanoarchitectures have been candidate for various applications in biology, nanotechnology, and medicine, i.e. nanomedicine. DNA nanotubes have also been employed as nanowires loaded with various nanoparticle types and suggested them as electrical conductors [34, 35]. DNA nanotubes could also be applied in drug delivery systems because of their high capacity for carrying more cargoes [5, 8]. The nanotubes could also be substituted with carbon nanotubes due to their compatibilities with the life systems . Therefore, design and development of a cost benefit procedure for simply fabrication of DNA nanotubes would be essential. It seems fabrication of DNA nanotubes via ligation of DNA nanotemplates with designed sticky ends could be a simplified procedure for providing stabilized DNA nanotubes with the minimum staples when assisted with a ligase treatment. This study also demonstrated the role of STM in high-resolution imaging of DNA nanotubes in air condition with no use of the vacuumed chamber commonly required in electron microscopes.
P. Gill conceived and designed the experiments. S-M. Mousavi-Khattat and A. Rafati equally performed the experiments. P. Gill analysed the data. P. Gill contributed reagents/materials/analysis tools. S-M. Mousavi-Khattat and P. Gill wrote the paper.
This study was supported by Vice Chancellor of Research and Technology, Golestan University of Medical Sciences Grant no. 352438. Also, Iran Nanotechnology Initiative (INI) supported this project partially. The authors thank Batool Yousofi for her collaboration with this study.
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