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

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

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

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

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

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

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)

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

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

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

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

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

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

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

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

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