1. Sunmin Ahn Journal Club Presentation October 23, 2006
Folding DNA to Create Nanoscale Shapes and Patterns Paul W. K. Rothemund Nature, V440, , 2006
Sunmin Ahn
Journal Club Presentation
October 23, 2006

2. Outline Introduction Review of DNA structure Designing DNA origami
Folding with viral genome
Patterning
Conclusion

3. Introduction Parallel synthesis of nanostructures
Building DNA patterns and shapes with a long ssDNA and a bunch of staple strands
One pot self assembly

4. DNA Structure

5. Designing Pattern 1. Generation of block diagram
- Manual design
1. Generation of block diagram
2. Generation of a folding path
- raster fill pattern must be hand designed

6. Designing Pattern 3. Generation of a first pass design
- Computer aided
3. Generation of a first pass design
- raster fill pattern must be hand designed
- no bases left unpaired
- single phosphate from each backbone occurs in the gap
- small angle bending does not affect the width of DNA origami

7. Designing Pattern 4. Refinement of the helical domain length
- Computer aided
4. Refinement of the helical domain length
- to minimize strain in design
- twist of scaffold calculated and scaffold x-over strains are balanced by a single bp change
- periodic x-overs of staples are arranged with glide symmetry
 minor groove faces alternating directions in alternating columns

8. Designing Pattern 5. Breaking and merging of strands - Computer aided
- pairs of adjacent staples are merged to yield fewer, longer staples
- merge patterns are not unique
- staggered merge strengthens seam

9. Designing Pattern 5. Breaking and merging of strands - Computer aided
- rectilinear merge

10. Folding viral genome
Circular genomic DNA from virus M13mp18 chosen as a scaffold
Naturally ssDNA 7249-nt long
For linear scaffold 73-nt region containing 20-bp stem hairpin was cut with BsrBI restriction enzyme
resulting 7167nt long linear strand
100X excess of staples and short (<25nt) remainder strands mixed with scaffold and annealed 95ºC to 20ºC in a PCR machine (< 2 hours)
Samples deposited on mica and imaged with AFM in tapping mode

11. Folding viral genome Square Rectangle linear scaffold 13% well formed
25% rectangular fragments
25% hourglass fragments
Rectangle
tests “bridged” seam
circular scaffold
90% well formed
1μm scale bars

12. Folding viral genome Star Smiley demonstrates certain arbitrary shape
linear and circular scaffold
11% and 63% well formed
higher % of well formed shapes with circular scaffold may be due to higher purity of the scaffold strand
Linear scaffold
Circular scaffold
100nm scale bars
Smiley
circular scaffold
need not be topological disc
90% well formed
narrow structures are difficult to form  provides “weak spot”
100nm scale bar

13. Folding viral genome Triangle from 3 rectangles
single covalent bond holding the scaffold together
less than 1% well formed
stacking
100nm scale bar
Triangle built from 3 trapezoids
circular scaffold
88% well formed with bridging staples
55% well formed without bridging staples
100nm scale bar

14. Stacking Interaction between blunt end helices cause stacking
Staple strands on the edge may be removed (B)
Addition of 4T hairpin loops (F)
Addition of 4T tails on staples that has ends on the edge of the shape (D)
Stacked rectangles
Staple strand on the edge removed F C D
Normal amount of aggregation (Smileys)
Addition of 4T tails
1μm scale bars

15. Defects and Damages
100nm scale bars

16. Stoichiometry
In most experiments 100~300 fold excess over scaffold was used
10 fold excess is safe, but not a fundamental requirement
2-fold excess may be used
1μm scale bars

17. Patterning

18. Patterning Binary patterning “1” – 3nm above mica surface
1μm scale bars

19. Patterning
Infinite periodic structures are made using extended staples
Stoichiometry becomes very important
~30 Megadalton structure (individual origami ~4megadalton)
100nm scale bars

20. Difficulties Blunt end stacking Down hairpin loops
But mostly AFM imaging!!!

21. What about 2º Structures?
Lowest E folds calculated
Strong structure
Weak structure
Average kcal/mole
Random 6000 base sequence generated with same base composition as M13mp18
- Similar 2º structure
- Average free E kcal/mole

22. How does it work? Strand invasion Excess of staples
Cooperative effects
Designs that doesn’t allow staples to bind to each other

23. Conclusion Quantitative and statistical analysis
Better imaging technique should be implemented
DNA nonostructure patterning may be used as templates for programmed molecular arrays
Protein arrays
nanowires