1. Presented by Pardeep Dhillon and Ehsan Fadaei
Design and self-assembly of two-dimensional DNA crystals Erik Winfree, Furong Liu, Lisa A. Wenzler & Nadrian C. Seeman
Presented by Pardeep Dhillon and Ehsan Fadaei

2. Purpose
How to control detailed structure of matter on the finest possible scale
Need for a rigid design component with predictable and controllable interactions which led to the idea of the antiparallel DNA double-crossover motif

3. Introduction
Double-crossover (DX) molecules are analogues of intermediates in meiosis
They contain “sticky ends” in order to combine them into a 2D periodic lattice
DX molecules can act as Wang tiles (rectangular tiles with programmable interactions) which self-assemble to perform desired computations

4. Wang Tiles
These subunits can only be placed next to each other if their edges (sticky ends) are identical
2 tiles, A & B, make a striped lattice
4 tiles, A, B, C & D, make a striped lattice with double the period
Overall, these systems self-assemble in solution into 2D crystals that have a defined subunit structure

5. Model Structures for DAO and DAE units
Antiparallel DX motif contains 2 juxtaposed immobile 4-arm junctions with non-cross-over strands being antiparallel to each other
Only 2 of 5 DX motifs are stable → DAO or DAE
DAO (double crossover, antiparallel, odd spacing)
Has 4 strands and 3 half-turns per crossover point
DAE (double crossover, antiparallel, even spacing)
Has 5 strands and 4 Half-turns per crossover point

6. Differences in DAO-E and DAE-O systems
Used the 2 systems to make a 2-unit lattice each separately
DAE-O design involves 2 small nicked circular strands, 2 horizontal and 2 vertical strands
The horizontal and vertical strands can act as reporters of self-assembly on a gel
DAO-E has the advantage of using simple, 4 vertical strand DX units

7. Sequences of DX units
DAO-E DAE-O
Illustration of sequences of the DX subunits showing the sticky ends
B^ subunit contains 2 hairpin-terminated bulged 3-arm junctions
This feature allows for visualization on Atomic Force Microscopy(AFM)

8. Analysis of Lattice Assembly
T4 polynucleotide kinase used to phosphorylate strands with 32P
After annealing, added T4 DNA ligase to link subunits covalently
Samples are performed on denaturing gel
Odd lanes (3-9) contain exonuclease I and III to see if any circular products are present

9. Gel Image Results
Gel image shows that sticky ends of A units have affinity for sticky ends of B units
Each subunit in the DAE-O design contains 4 continuous strands and one circular strand
Enzymatic ligation of lattices with T4 DNA Ligase produced long covalent DNA strands
Direct physical observation (ie. AFM) is necessary to confirm lattice assembly

10. Atomic Force Microscopy (AFM)
AFM has a microscale cantilever with a sharp tip that scans the surface of the sample
A laser is reflected against the cantilever and any deflection is measured by an array of photodiodes
To make sure the tip does not damage sample, it uses a feedback mechanism that measures surface-tip interactions on a scale of nanoNetwons

11. AFM Procedure 2 Methods to visualize the 2D lattice by AFM
Incorporated 2 hairpin structures OR
Chemical labeling via biotin-streptavidin-nanogold particles
DAE-O B subunit was labeled with a 5’ biotin group
After AB assembly, added 1.4nm nanogold-steptavidin
Imaged sample by AFM

12. AFM Images a) DAO-E AB lattice b,c) DAO-E AB^ lattice
d) DAE-O AB lattice
e,f) DAE-O AB^ lattice
DAE-O AB^ lattice stripes have 33±3 nm periodicity
DAO-E AB^ lattice stripes have 25±2 nm periodicity

13. AFM Images a,b,c) DAO-E AB^ lattice d) DAE-O AB lattice
B subunit labeled with biotin-streptavidin-nanogold
e,f) DAE-O ABCD^ lattice
DAE-O ABCD^ lattice stripes have 66±5 nm periodicity

14. Summary 2 types of stable lattice designs → DAE-O and DAO-E
A and B subunits can self-assemble together via specific sticky ends to make the lattice
They can’t anneal with themselves
Incorporation of hairpin structures or biotin- streptavidin-nanogold labeling allows for visualization of periodicity by AFM

15. Future Directions
Self-assembly is becoming recognized as a route to nanotechnology such as biochips
It should be possible to control the structure with chemical groups, catalysts, enzymes, nanoclusters, DNA enzymes, etc…
It may be possible to make the 2D lattice into 3D
Improve methods for error reduction and purification

16. Thank You!