DESIGN AND SELF-ASSEMBLY OF TWO-DIMENSIONAL DNA CRYSTALSERIK WINFREE, FURONG LIU, LISA A. WENZLER & NADRIAN C. SEEMAN

Presented by Pardeep Dhillon and Ehsan Fadaei

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Purpose2

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

Introduction3

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

Wang Tiles4

• 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

Model Structures for DAO and DAE units

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• 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

Differences in DAO-E and DAE-O systems

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•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

Sequences of DX units7

•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)

DAO-E DAE-O

Analysis of Lattice Assembly

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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

Gel Image Results9

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

Atomic Force Microscopy (AFM)

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•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

AFM Procedure11

2 Methods to visualize the 2D lattice by AFM1. Incorporated 2 hairpin structuresOR2. 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

AFM Images12

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

AFM Images13

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

Summary14

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

Future Directions15

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

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