View
221
Download
1
Tags:
Embed Size (px)
Citation preview
Folding DNA to Create Nanoscale Shapes and PatternsPaul W. K. RothemundNature, V440, 297-302, 2006
Sunmin Ahn
Journal Club Presentation
October 23, 2006
Outline
• Introduction • Review of DNA structure • Designing DNA origami• Folding with viral genome• Patterning• Conclusion
Introduction
• Parallel synthesis of nanostructures• Building DNA patterns and shapes with a long
ssDNA and a bunch of staple strands• One pot self assembly
DNA Structure
Designing Pattern
1. Generation of block diagram 2. Generation of a folding path- raster fill pattern must be hand designed
- Manual design
Designing Pattern
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
- Computer aided
Designing Pattern
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
- Computer aided
Designing Pattern
5. Breaking and merging of strands- pairs of adjacent staples are merged to yield fewer, longer staples
- merge patterns are not unique
- staggered merge strengthens seam
- Computer aided
Designing Pattern
5. Breaking and merging of strands- rectilinear merge
- Computer aided
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
Folding viral genome
Square- linear scaffold- 13% well formed- 25% rectangular fragments- 25% hourglass fragments
Rectangle - tests “bridged” seam- circular scaffold- 90% well formed
1μm scale bars
Folding viral genome
Star- 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
Smiley- circular scaffold- need not be topological disc- 90% well formed- narrow structures are difficult to form provides “weak spot” 100nm scale bar
100nm scale barsLinear scaffold Circular scaffold
Folding viral genome
Triangle from 3 rectangles- single covalent bond holding the scaffold together- less than 1% well formed- stacking
Triangle built from 3 trapezoids
- circular scaffold- 88% well formed with bridging staples- 55% well formed without bridging staples 100nm scale bar
100nm scale bar
Stacking
Normal amount of aggregation (Smileys)
Addition of 4T tails
Stacked rectangles Staple strand on the edge removed
1μm scale bars
1. Staple strands on the edge may be removed (B)
2. Addition of 4T hairpin loops (F)
3. Addition of 4T tails on staples that has ends on the edge of the shape (D)
A B
C D
F
Interaction between blunt end helices cause stacking
Defects and Damages
100nm scale bars
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
Patterning
Patterning
Binary patterning“1” – 3nm above mica surface
“0” – 1.5nm above mica surface
1μm scale bars
Patterning
Infinite periodic structures are made using extended staples• Stoichiometry becomes very important• ~30 Megadalton structure (individual origami ~4megadalton)
100nm scale bars
Difficulties
- Blunt end stacking- Down hairpin loops- But mostly AFM imaging!!!
What about 2º Structures?
• Lowest E folds calculated
Strong structure Weak structure
• Average -965+-37kcal/mole• Random 6000 base sequence generated with same base composition as M13mp18
- Similar 2º structure- Average free E -867 +- 13kcal/mole
How does it work?
1. Strand invasion
2. Excess of staples
3. Cooperative effects
4. Designs that doesn’t allow staples to bind to each other
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