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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

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Presented by Pardeep Dhillon and Ehsan Fadaei

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  1. 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

  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!

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