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