An interesting concept of DNA origami and a brief description of its application in drug delivery

1. SNEHAL MENON
Integrated M.Tech-PhD

2. INTRODUCTION
• The field of structural DNA nanotechnology started around 30
years ago when Ned Seeman* performed pioneering
research with DNA junctions and lattices
• The key player in the fast development of DNA
nanotechnology was the invention of DNA origami in 2006
• The DNA origami method is based on folding a long single-
stranded ‘DNA scaffold strand’ into a customized shape with
a set of short synthetic strands that act as ‘staples’ to bind
the overall structure together.
*Seeman, N.C. (1982) Nucleic acid junctions and lattices. J. Theor. Biol. 99, 237–247
M.C. Escher’s “Depth”

3. DESIGN OF SCAFFOLDED DNA ORIGAMI
1. Generation of a block diagram 2. Generation of a folding path
Five phases: two manual design steps and three passes of the program

4. 3. Design of staple strands

5. 4. Refinement of the helical domain lengths
• Crossovers along the edges of the shape, in particular,
must be adjusted to minimize strain
• The twist of scaffold crossovers is calculated and their
position is changed (typically by a single bp) to minimize
strain; staple sequences are recomputed accordingly
5. Breaking and merging of strands

6. • One pot’ reaction:
• ~200–250 staple and remainder strands + scaffold
• Annealed from 95⁰C to 20⁰C in a PCR machine <2 h
• AFM imaging

7. DESIGN OF THREE-DIMENSIONAL DNA ORIGAMI

8. caDNAno
• Slice panel: provides x–y cross-section
view of the honeycomb helix lattice with
helices represented as circles
• Path panel: used for nucleotide-level
editing of scaffold and staple-path
connectivity
• Render panel: provides a real-time, 3D
cylinder model for visualizing the shape
as it is constructed

9. • Short stretches of
scaffold are inserted
into the Path panel as
helices are added via
the Slice panel
• The Path panel editing
tools are used to stitch
together a continuous
scaffold path.
• The ‘auto-staple button’ is
used to generate a default
set of continuous staple
paths, including crossovers.

10. • Staple paths are created wherever scaffold is present, according to an algorithm that
follows the aforementioned rules for crossover spacing
• After all staples are edited into a satisfactory arrangement, the scaffold path is populated
with a DNA sequence using the ‘Add Sequence’ tool
• Several default sequences are provided, or the user can input his or her own
• Additionally, a 3D model can be exported in X3D format, with double helices represented
as cylinders

11. ROLE OF DNA ORIGAMI IN
CANCER THERAPY

12. DNA ORIGAMI AS AN IN VIVO DRUG DELIVERY VEHICLE FOR
CANCER THERAPY
• DNA origami nanostructures as a biocompatible drug carrier system: Qiao Jiang, et al 2012
• Doxorubicin origami structures were effectively internalized by tumor cells
• The triangle-shaped DNA origami exhibits optimal tumor passive targeting accumulation: Qian
Zhang, et al 2014
• M13 DNA, triangle, tube, and square DNA origami nanostructures were characterized

13. Biodistribution in a subcutaneous breast tumor model
• M13 DNA and three DNA origami shapes incorporated with
equivalent doses quantum dots
• Triangle DNA origami nanostructure exhibited the optimal
accumulation at the tumor regions compared with square DNA
origami and tube DNA origami
• Fluorescence signals: Triangle > Square > Tube
• In contrast, the fluorescence signal of the free QD and QD-M13
DNA at tumor sites was weak

14. • At 24 h postinjection the same mice were sacrificed, and the tumor and major organs (brain,
tumor, liver, kidney, spleen, lung, and heart) were collected
• The ex vivo fluorescence imaging results showed that QD-tube origami and QD-square origami was
distributed not only in tumor but also in liver and kidney
• The QD-triangle origami was mainly accumulated in tumor and slightly in liver
• The ex vivo result was consistent with the in vivo biodistribution, suggesting optimal shape-
dependent uptake in the tumor tissues

15. Drug loading and distribution of Doxorubicin within tumor tissues
• Triangle DNA origami was loaded with doxorubicin and the accumulation of DOX and DOX/origami
at the tumor sites was observed by fluorescence imaging
• After administering saline, DOX and DOX/origami, the tumor tissues were collected at 24 h
• The fluorescence showed that there was relatively more doxorubicin at the tumor sites in the
DOX/origami-treated group compared with the DOX alone group
• The drug delivered by DNA origami carriers was mainly distributed surrounding blood vessels of
tumor regions
• By combining passive accumulation with slow drug release in vitro, DOX/origami holds the
potential to reduce the nonspecific distribution of doxorubicin during the in vivo delivery
process, inducing controlled drug release in the tumor region

16. CONCLUSION
• With the advent of DNA origami, the transition from platonic structures to nano-objects capable
of performing predefined tasks and their sophisticated implementations has been extremely fast
• The advantages of using DNA-based nanostructures in therapeutics over the other accessible
nanosized systems go beyond their intrinsic biocompatibility and biodegradability
• The most important aspect is their modularity – the size of an object and the positions of
modifications can be precisely controlled at nanometer scale and, moreover, the shape and the
flexibility of the object can be fine-tuned
• These superior and adjustable properties facilitate straightforward characterization of the DNA
nanostructures (labeling/bioimaging) and the engineering of targeting and releasing features for
delivery purposes
• Thus, the current and imminent DNA nanoassemblies will have a huge impact on advanced
health sciences and clinical chemistry

17. OTHER APPLICATIONS
• DNA structures for small interfering RNA (siRNA) delivery
• DNA structures for CpG triggered immunostimulation
• Rectangular DNA origamis coated with virus capsid proteins (CPs) for efficient cellular
delivery
• A virus-inspired membrane-encapsulated spherical DNA origami vehicle for decreasing
immune activation and enhancing pharmacokinetic bioavailability
• A DNA origami that can form an ion channel in a lipid membrane
• The structures that can be inserted into cell membranes, and the pores can kill cancer cells.

18. REFERENCES
1. DNA Nanostructures as Smart Drug-Delivery Vehicles and Molecular Devices; Kostiainen et al;
Trends in Biotechnology, October 2015, Vol. 33, No. 10, 586-594.
2. Folding DNA to create nanoscale shapes and patterns, Paul W. K. Rothemund, Vol 440, 16 March
2006, doi:101038/nature04586, 297-302
3. DNA nanotubes as intracellular delivery vehicles in vivo, Sabine Sellner et al, Biomaterials 53
(2015) 453-463
4. Nucleic Acid Based Molecular Devices, Yamuna Krishnan and Friedrich C. Simmel, Angewandte
Chemie, Int. Ed. 2011, 50, 3124 – 3156
5. Self-assembly of DNA into nanoscale three-dimensional shapes, Shawn M. Douglas et al, Vol 459,
May 2009, doi:101038/nature08016, 414-418
6. Rapid prototyping of 3D DNA-origami shapes with caDNAno, Shawn M. Douglas, Nucleic Acids
Research, June 2009, Vol. 37, No. 15, 5001–5006
7. DNA Origami as an In Vivo Drug Delivery Vehicle for Cancer Therapy, Qian Zhang et al, American
Chemical Society, VOL. 8 , NO. 7 , 2014, 6633–6643