Question to be addressed Electroporation, lipid transfection, and biolitic particle delivery systems have been used to deliver genetic material into cells but either are unsuitable for delivery to mammals; or there is lack of success in delivering the DNA

Abstract

Introduction:

DNA nanotechnology is the study of DNA at nanometre scale. It’s used to design models using DNA origami (a synthetic folding method) to enhance drug delivery. This is achieved by encapsulating Active Pharmaceutical Ingredient (API) inside the design for targeted delivery.

Background:

DNA Origami is utilised by constructing synthetic nanoparticles using computer-aided-software (e.g. CaDNAno). Due to the strict base-pair ruling in DNA, its self-assembly and folding ability has proven many potential outcomes that has been conducted in vitro.

Challenges:

DNA nanotechnology has its uncertainty, due to its uncommon non-biological use of designing DNA origami structures in nature and the lack of its biological relevance because of its limited applications.

The study:

The study will aim to design a nanoparticle using the plasmid ‘pCS2+8CmCherry’, which contains a short sequence. The design will potentially enhance self-assembly and folding, which has been challenging previously.

The results identified a design within the limiting size for nanoparticles, the design was strengthened using numerous staple strands. It contained breaks in design which can be used to encapsulate API.

Conclusions

DNA nanotechnology will still need extensive research, however in this design new factors have been researched. This includes using a smaller plasmid that contains a fluorescent protein, which can enhance identification for successful drug delivery.

Nevertheless, more research is required around this study, as its potential still requires establishing. The study contains designs constructed as a blueprint which can be tested using the protocol for its relevance.

Introduction

Gene therapy has grown its potential in clinical benefits; however it has impacted on its development in medicine. It has been recognised initially to correct and prevent prognosis of genetic disease. (Fischer, 2016)

Gene therapy involves the delivery of genetic material to target tissue or cells to achieve a therapeutic end-point. Gene therapy has potential for treatment for viruses, cancers, and genetic disorders. DNA vaccination is a technique used in protecting against diseases by injecting genetically engineered DNA plasmid into target cells of the body to produce antigenic proteins; a promising technique in comparison to other interventions. However, it has shown disappointing potency of DNA vaccines, that have been experimented successfully in pre-clinical models in vitro but in comparison to clinical trials in vivo not as effective. (Donnelly, Wahren and Liu, 2005)

DNA Nanotechnology is the synthetic design and study of DNA applications used for diagnosis, control and treatment of diseases. DNA has been used to design nanostructures for delivery of API to chosen cells, this helps prevent the damage of normal cells. The benefits of its base pairing forms rigidity in the design of nanoparticles, easy formation of varied length of strands allows construction of 2D and 3D shapes with adaptable nontoxic nanoparticles. (Chakraborty, Roy and Mondal, 2016)

DNA nanotechnology is the study of DNA at a nanometre scale, the understanding of this study includes; it’s self-assembly function and base pairing, physical and chemical properties of the nanostructures. Structural nanotechnology focuses on synthesising nucleic acid complex, dynamic technology focus on the ability to reconfigure structures based on chemical and physical stimuli. Prior to DNA origami, tile-based structures and dynamic assembly was used for DNA structural designs. (Dietz, Douglas and Shih, 2009)

Structural Properties of DNA enables it to be used effectively for other purposes, the complimentary base pairing helps utilize DNA to be used as nanoparticles which is designed via DNA origami. Nadine Seaman first identified the principles of DNA nanotechnology in 1980s, with the motivation to create 3D DNA structure. (Seeman, 2005)

DNA origami has enabled the construction of scaffold structures that have self-assembling ability; this can be used for arrangement of different functional nanomaterial to produce complex devices. Seeman created various DNA strategies using self-assembly technique and played an important role in the basic concepts of DNA nanotechnology. DNA origami has attracted attention since invention; it can programme DNA assembly in nanotechnology which enables design of 2D and 3D nanostructures with functionality. (Endo, Yang and Sugiyama, 2013)

In 2006, Paul Rothemund introduced and demonstrated the scaffolded DNA origami technique. The technique revealed single long stranded DNA being utilised as a Scaffold DNA to fold and support short stranded DNA known as staples. The use of DNA origami technique has been successful in nanoscale folding, due to the interactions between complementary base pairs forming rigid stable structures. (Seeman, 2007; Udomprasert and Kangsamaksin, 2017)

DNA origami is the folding of DNA to create nanoscale structures in 2D or 3D shapes. It has been discovered that DNA can be used for more than just its natural biological use. It’s useful as construction material because of it’s self-assembly for building structural designs. The technique has become successful for its potential application in nanotechnology, as construction of large material in short time was possible. DNA origami developed potential 2D objects such as dolphins, nanotubes and later 3D structure (sphere, box) were made. (Udomprasert and Kangsamaksin, 2017)

The 3D DNA ‘box’ designed by Andersen, et al (2009) helped extend the use of DNA origami, as this box can open by the presence of DNA keys. The DNA box was designed using circular single stranded DNA from a viral bacteriophage m13, this was mixed with staple sequences and heated to design the self-assembled DNA box. DNA Nanotechnology is being used in non-biological forms, and not as carrier of genetic material. The structural architecture of DNA is used to design different technological outputs and is chosen because it is non-toxic, naturally present in cells, has strict base-pairing and has a natural ability to self-assemble. (Seeman, 2005)

Historically, DNA nanotechnology has its uncertainty due to its uncommon non-biological use of designing DNA origami structures in nature and the lack of its biological relevance it has had little applications. The issue is resolving misfolded structures to produce chosen structures. Misfolded structure kinetically decreases system output and yield, these are resolved and understood by examining assembly pathways and kinetics for increased successful folding. This will increase efficiency of folding pathways by understanding reversing misfolded bonds and directing structures to the anticipated design. (Dunn et al., 2015)

Plasmids are typically found as circular DNA in bacteria and other organisms, which carry genetic information for survival. Vectors are plasmids that are artificially used in molecular cloning gene therapy. Chakraborty et al, (2016), used single stranded bacteriophage DNA (M13) as the scaffold structure and used various designs of staples to construct different DNA origami structures.

Use of non-viral nanoparticles for DNA delivery has become more desirable, which are possible synthetic systems that work similarly to viral vectors. Gokirmark et al (2012), cloned a species of sea urchin; Stronglycentrotus purpuratus. The production formed variant vector ‘pCS2+8’, with modifications of N and C terminal variants for the addition of fluorescent proteins. Furthermore, the fluorescent protein ‘mCherry’ is notably more photostable and has greater intrinsic brightness than other fluorescent proteins. (Day and Davidson, 2009).

However, Gokirmark et al (2012), stated that the variants of the vector set have many significances of the proteins in development and disease but little knowledge regarding their phylogenetic origins and functional diversity. The plasmid is available on ‘addgene’ repository, pCS2+8CmCherry (Plasmid #34935).

CaDNAno is an open-source software that helps aid the design of DNA sequences being folded into 2D and 3D structures. Douglas, et al (2009) followed the techniques of Paul Rothemund’s 2D DNA origami structures and demonstrated 3D DNA structures.

This study will consist of designing a nanoparticle using caDNAno, containing the plasmid sequence of ‘pCS2+8CmCherry’ and identifying the potential behind the design. Furthermore, Autodesk Maya 2015, will be used to render the model and understand the potential architecture of the design, and its possible relevance to DNA origami.

Materials and Methods

The Design

· CaDNAno open-source software

· Autodesk Maya 2015

· Addgene plasmid depository

· pCS2+8CmCherry (addgene plasmid#:34935) sequence

· Microsoft Excel

· Oligocalculator

· Bioinformatics reverse complement

The plasmid was chosen during the proposal of the research project; the anticipated plasmid was chosen from addgene, a plasmid depository. This provides easy accessibility for researchers to conduct the experiment. The plasmid is a non-viral vector derived from a Sea Urchin species, Stronglycentrotus purpuratus. The plasmid has been developed via many variants and is the pCS2+8CmCherry (addgene plasmid#:34935). (Gokirmak et al., 2012)

All the work carried out on computer was using CaDNAno and Autodesk Maya 2015. CaDNAno is a computer aided design tool, the software has been used previously, to design custom shaped 2D and 3D origami shapes. CaDNAno uses two methods of forming pleated layers of helices; Honeycomb and Square lattice. The method chosen for this design was the square lattice as it can form compact 3D origami designs. By providing a densely packed construction and natural framework for rectangles, these foundations increase the versatility and scope of 3D origami shapes for DNA nanotechnology. (Ke et al., 2009)

As the plasmid size of ‘pCS2+8CmCherry’ is 4848 base-pairs, the square lattice was designed using 35 helices, in a 6x6 format, the order of helices represents the order of folding. The base-pair length was increased to 127 from a default of 63, both forward and reverse scaffold was produced on one palette.

The forward scaffold helices were joined via crossovers to increase folding of the shape, the same procedure followed on the reverse scaffold. The forward scaffold was coloured in blue and the reverse scaffold in red.

Once the desired scaffolds were completed, the ‘autostaple’ button was selected to add staple strands that interlinked both scaffolds. Autostaple provides the staples to be added automatically, but staples are too long and may not be in desired order.

The ‘autobreak’ button was selected to change parameters of staple strands. The parameters set was; target length of 48, minimum length of 24, maximum length of 90 and minimum distance to crossover at 4. This shortened the length of the staples which were rearranged to desired locations, to increase interlinking between both scaffold and ability of rehybridization. The staples were coloured in green, black and purple. Staples in green represented connection between helices on forward scaffold, black staples were connection between forward and reverse scaffold, and purple staples represented connection between helices on reverse scaffold.

After repositioning the staple order, the forward plasmid sequence was exported from ‘addgene’ and inserted onto forward scaffold using ‘add sequence’ tab on Autodesk Maya. The sequence was then inputted into a bioinformatics reverse complement which reverses the forward sequence; this reversed sequence is inputted into the reverse scaffold.

Staple strands and forward/reverse scaffold were viewed, the base pairs were complementary of each other, the list of staples were exported into Microsoft excel.

Furthermore, Autocad Maya 2015 is a tool used for architectural design; Maya was used to edit the model so clear visualization of the nanoparticle is possible. This was carried out by using rendering tool to increase the vibrant colours of the shape, and render view was used to produce images of the frame in JPEG and TIFF file, with a 300dpi resolution.

As the limiting size of the shape is required the dimensions (height, width, length) was measured on Autodesk Maya using the measuring tool. These dimensions were measured in centimeters (cm) as the use of Autodesk Maya has been used in architectural building designs, therefore the units were changed to nanometers (nm) as Maya is a tool that produces a blueprint for the design. The conversion from cm to nm is explained in the results section; design meeting limiting size.

Protocol

· pCS2+8CmCherry Addgene (plasmid cat#34935)

· HPLC purified staple list

· Polyethylene glycol (PEG)

· Precipitation buffer

· Ultracentrifuge

· Agarose gel electrophoresis system

· TEM-imaging

· AFM-imaging

1. The plasmid sequence can be purchased from Addgene (plasmid cat#34935), and the HPLC purified oligonucleotides (staple strands) using staple list obtained from design.

2. Preparation of Plasmid sequence ‘pCS2+8CmCherry’ is carried out using Polyethylene glycol (PEG), it is efficient in separating folded designs from excess of staple strands. Precipitation using PEG is recognised as an effective method in purifying and concentrating the DNA plasmid. (Wagenbauer et al., 2017)

3. Preparation of self-assembled design is carried out using stoichiometric quantities of plasmid sequence and desired buffer solution, with excess of purified oligonucleotides as staples.

4. Add a 1:1 ratio of unpurified sample (plasmid sequence) to PEG buffer solution, thoroughly mix to achieve homogenous assembled products and buffer, the specimen is separated via ultracentrifugation.

5. The supernatant is removed, and the pellet is dissolved in a buffer, to purify the sample, further purification methods can be carried out to remove PEG residue. (Stahl et al., 2014)

6. The next stage is annealing with oligonucleotides, the melting point of the plasmid calculated is 86°C, and this is taken into consideration to identify optimum annealing conditions.

7. The samples are then processed via purified agarose gel electrophoresis to identify characterisation of the design and rehybridization properties. Mechanism of design is identified. (Marras et al., 2016)

8. TEM is used to characterise the model projection in different orientation, (XZ, YZ and XY projections), for a 3D perspective view. The AFM imaging will enable the fluorescence imaging of the plasmid and its ‘mCherry’ component. (Ke et al., 2012)

Results

Use of CaDNAno to design a nanoparticle

The design was developed using open-source software CaDNAno, this helped as a graphical interface that aids the design of DNA origami to produce 2D and 3D structures containing DNA sequences. It has helped reduce the effort to design DNA origami, by minimizing errors when carrying out tasks, having an efficient output of effort and completion of design. (Douglas et al., 2009)

Figure 1: The design of a square lattice on CaDNAno using 35 helices.

To determine whether the chosen plasmid fits the design constructed on caDNAno, the chosen plasmid had a length of 4848bp (empty backbone 4095bp). The design consisted of tightly packed square lattice structure, the default base-pair length along x-axis was set to 63 base pairs. A multiple of 32 was required, the helices was extended to a base-pair of 127. The scaffold size calculated to a total of 4444bp (35 helices x 127bp).

Nevertheless, this was smaller than the length of the plasmid, and there is difficulty in understanding what software does with excess plasmid length. As each helix contained 127bp, which majority was visually understood to complementary pair with staple strands. The remaining plasmid sequences are used for the front and end unpaired scaffold crossovers, and crossovers in the middle. (Douglas et al., 2009)

Figure 2: An illustration of the design, unpaired forward, middle and end scaffold crossovers is circled. No plasmid sequence is shown this is the location where the excess plasmid is used.

To be able rehybridization of the plasmid after delivery into cells, the design illustrates the interweaving of both the forward and reverse scaffold. This will increase the rigidity of the shape the plasmid forms, preventing it degrading in vivo, as it is densely packed with dsDNA, which enhances folding.

Figure 3: Illustrates the interweaving of both forward and reverse scaffold.

To design a structure that can encapsulate the API, this was carried out by further increasing the interlinking between forward and reverse scaffold, increasing the potential to fold. The design will fold and form a cuboidal shape that has a cavity space.

Figure 4: This figure shows the forward scaffold (blue) and reverse scaffold (red), with increased interweaving to strengthen the model and increase folding.

Once both scaffolds were designed on a single palette, it was autostapled to add staple strands to interlink both the scaffolds together. Although this method is random allocation of staples with different lengths, the autobreak function was used to change the parameters of staple strands.

Figure 5: Shows autostaples added randomly, this is further edited manually.

What does the positioning of the oligonucleotides determine? The positioning of the staples is manually altered, first by selecting ‘autobreak’ to change parameters of staple strands. The parameters set was; target length of 48, minimum length of 24, maximum length of 90 and minimum distance to crossover at 4. This is performed to alter arrangement of staples, to increase interlinking between both scaffold and enhancement of rehybridization.

Figure 6: Illustration of autobreak function, it has removed thick long staple strands.

What does the design show, and what has been achieved? The design shows the Forward scaffold in blue and Reverse scaffold in Red. The staples are colored in green, black and purple. Green staples are cross-overs between different helices on the forward scaffold, Black staples are cross-overs between helices on the Forward and Reverse scaffold, and Purple staples are cross-overs between different helices of Reverse scaffold. This is to prevent degradation of the design; therefore, number of staples are increased. This will enhance the possibility of targeted delivery.

Figure 7: The design has different colours so the model can be visualized in 3D.