DESIGN AND SELF-ASSEMBLY OF TWO-DIMENSIONAL DNA CRYSTALS

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Group presentation for the 4th year Nucleic Acid course at SFU. The presentation is about a scientific paper which discusses the nature of two-dimensional DNA crystals. The authors of the paper include Erik Winfree, Furong Liu, Lisa A. Wenzler & Nadrian C. Seeman.

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DESIGN AND SELF-ASSEMBLY OF TWO-DIMENSIONAL DNA CRYSTALS

  1. 1. 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. 2. Purpose <ul><li>How to control detailed structure of matter on the finest possible scale </li></ul><ul><ul><li>Need for a rigid design component with predictable and controllable interactions which led to the idea of the antiparallel DNA double-crossover motif </li></ul></ul>
  3. 3. Introduction <ul><li>Double-crossover (DX) molecules are analogues of intermediates in meiosis </li></ul><ul><li>They contain “sticky ends” in order to combine them into a 2D periodic lattice </li></ul><ul><li>DX molecules can act as Wang tiles (rectangular tiles with programmable interactions) which self-assemble to perform desired computations </li></ul>
  4. 4. Wang Tiles <ul><li>These subunits can only be placed next to each other if their edges (sticky ends) are identical </li></ul><ul><li>2 tiles, A & B, make a striped lattice </li></ul><ul><li>4 tiles, A, B, C & D, make a striped lattice with double the period </li></ul><ul><li>Overall, these systems self-assemble in solution into 2D crystals that have a defined subunit structure </li></ul>
  5. 5. Model Structures for DAO and DAE units <ul><li>Antiparallel DX motif contains 2 juxtaposed immobile 4-arm junctions with non-cross-over strands being antiparallel to each other </li></ul><ul><li>Only 2 of 5 DX motifs are stable -> DAO or DAE </li></ul><ul><ul><li>DAO ( d ouble crossover, a ntiparallel, o dd spacing) </li></ul></ul><ul><ul><ul><li>Has 4 strands and 3 half-turns per crossover point </li></ul></ul></ul><ul><ul><li>DAE ( d ouble crossover, a ntiparallel, e ven spacing) </li></ul></ul><ul><ul><ul><li>Has 5 strands and 4 Half-turns per crossover point </li></ul></ul></ul>
  6. 6. Differences in DAO-E and DAE-O systems <ul><li>Used the 2 systems to make a 2-unit lattice each separately </li></ul><ul><li>DAE-O design involves 2 small nicked circular strands, 2 horizontal and 2 vertical strands </li></ul><ul><ul><li>The horizontal and vertical strands can act as reporters of self-assembly on a gel </li></ul></ul><ul><li>DAO-E has the advantage of using simple, 4 vertical strand DX units </li></ul>
  7. 7. Sequences of DX units <ul><li>Illustration of sequences of the DX subunits showing the sticky ends </li></ul><ul><li>B^ subunit contains 2 hairpin-terminated bulged 3-arm junctions </li></ul><ul><ul><li>This feature allows for visualization on Atomic Force Microscopy(AFM) </li></ul></ul>DAO-E DAE-O
  8. 8. Analysis of Lattice Assembly <ul><li>T4 polynucleotide kinase used to phosphorylate strands with 32 P </li></ul><ul><li>After annealing, added T4 DNA ligase to link subunits covalently </li></ul><ul><li>Samples are performed on denaturing gel </li></ul><ul><li>Odd lanes (3-9) contain exonuclease I and III to see if any circular products are present </li></ul>
  9. 9. Gel Image Results <ul><li>Gel image shows that sticky ends of A units have affinity for sticky ends of B units </li></ul><ul><li>Each subunit in the DAE-O design contains 4 continuous strands and one circular strand </li></ul><ul><li>Enzymatic ligation of lattices with T4 DNA Ligase produced long covalent DNA strands </li></ul><ul><li>Direct physical observation (ie. AFM) is necessary to confirm lattice assembly </li></ul>
  10. 10. Atomic Force Microscopy (AFM) <ul><li>AFM has a microscale cantilever with a sharp tip that scans the surface of the sample </li></ul><ul><li>A laser is reflected against the cantilever and any deflection is measured by an array of photodiodes </li></ul><ul><li>To make sure the tip does not damage sample, it uses a feedback mechanism that measures surface-tip interactions on a scale of nanoNetwons </li></ul>
  11. 11. AFM Procedure <ul><li>2 Methods to visualize the 2D lattice by AFM </li></ul><ul><ul><li>Incorporated 2 hairpin structures </li></ul></ul><ul><ul><li>OR </li></ul></ul><ul><ul><li>Chemical labeling via biotin-streptavidin-nanogold particles </li></ul></ul><ul><ul><ul><li>DAE-O B subunit was labeled with a 5’ biotin group </li></ul></ul></ul><ul><ul><ul><li>After AB assembly, added 1.4nm nanogold-steptavidin </li></ul></ul></ul><ul><ul><ul><li>Imaged sample by AFM </li></ul></ul></ul>
  12. 12. AFM Images <ul><li>a) DAO-E AB lattice </li></ul><ul><li>b,c) DAO-E AB ^ lattice </li></ul><ul><li>d) DAE-O AB lattice </li></ul><ul><li>e,f) DAE-O AB ^ lattice </li></ul><ul><li>DAE-O AB ^ lattice stripes have 33±3 nm periodicity </li></ul><ul><li>DAO-E AB ^ lattice stripes have 25±2 nm periodicity </li></ul>
  13. 13. AFM Images <ul><li>a,b,c) DAO-E AB ^ lattice </li></ul><ul><li>d) DAE-O AB lattice </li></ul><ul><ul><li>B subunit labeled with biotin-streptavidin-nanogold </li></ul></ul><ul><li>e,f) DAE-O ABCD ^ lattice </li></ul><ul><li>DAE-O ABCD ^ lattice stripes have 66±5 nm periodicity </li></ul>
  14. 14. Summary <ul><li>2 types of stable lattice designs -> DAE-O and DAO-E </li></ul><ul><li>A and B subunits can self-assemble together via specific sticky ends to make the lattice </li></ul><ul><ul><li>They can’t anneal with themselves </li></ul></ul><ul><li>Incorporation of hairpin structures or biotin-streptavidin-nanogold labeling allows for visualization of periodicity by AFM </li></ul>
  15. 15. Future Directions <ul><li>Self-assembly is becoming recognized as a route to nanotechnology such as biochips </li></ul><ul><li>It should be possible to control the structure with chemical groups, catalysts, enzymes, nanoclusters, DNA enzymes, etc… </li></ul><ul><li>It may be possible to make the 2D lattice into 3D </li></ul><ul><li>Improve methods for error reduction and purification </li></ul>

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