Sunmin Ahn Journal Club Presentation October 23, 2006 Folding DNA to Create Nanoscale Shapes and Patterns Paul W. K. Rothemund Nature, 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 - Manual design 1. Generation of block diagram 2. Generation of a folding path - raster fill pattern must be hand designed

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

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

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

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

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 Rectangle 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 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

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

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

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 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? Strand invasion Excess of staples Cooperative effects 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