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DESIGN AND SELF-ASSEMBLY OF TWO-DIMENSIONAL DNA CRYSTALS
 

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 ...

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 DESIGN AND SELF-ASSEMBLY OF TWO-DIMENSIONAL DNA CRYSTALS Presentation Transcript

    • 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 ( d ouble crossover, a ntiparallel, o dd spacing)
          • Has 4 strands and 3 half-turns per crossover point
        • DAE ( d ouble crossover, a ntiparallel, e ven 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
      • 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
      • T4 polynucleotide kinase used to phosphorylate strands with 32 P
      • 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