EXPERIMENT

MATERIALS

  • All staple DNA, anchor oligo DNA, glue DNA and gold nanoparticle were     purchased from Sigma-Aldrich Japan.
  • Single-stranded M13mp18 DNA were purchased from BioLabs Inc.

    • EXPERIMENT METHODS

    Method Ⅰ---Designing the DNA origami

    We designed our structure based on a previously established DNA origami (3) , and modified the strands a little. This modification was made to allow the AuNPs possible to attach to the sheet, and to allow the origami to connect to each other. The sequences of all the staple strands, the two different kinds of strands (glue strands) needed to connect the origami and the oligo DNA used to create the AuNP-anchor oligo DNA conjugate are available here.
      We designed the position of AuNPs attached to the sheet so that the distance between each gold particle becomes roughly the same.
      The tip of each triangle in the image below is where we attached the AuNP.

    Method Ⅱ---Synthesis of origami rectangle (1),(3)

    1. Dissolve each staple strands in 1×TAE buffer and prepare 0.2mM staple     solution.
    2. Mix all staple solution (2μl of each) to prepare staple-mix solution.
    3. Mix reagents for PCR as following order in micro tube(Table1.);

    Table1. Mix reagents for PCR
    Milli Q      3.9μl
    10×TAE       3μl
    12.5mM Magnesium acetate      7.5μl
    100nM Staple DNA solution      3.6μl
    20nM Scaffold DNA solution      12μl
    total      30μl

    4. Anneal sample as following conditions
    　　95℃ 5min
    　　↓
    　　95℃→20℃　gradually lower the annealing temperature
    　　 ( 1℃/min in 0.1℃ steps)
    5. Resulting solution contains DNA origami rectangles.
    サンプル

    MethodⅢ---Preparation of AuNP-anchor oligo DNA conjugates (2)

    1. Dissolve SH-modified ahchor oligo DNA in 0.072M DTT for 1h at room     temperature to remove the protective group.
    2. Purify anchor oligo DNA by using NAP-5 column.
    3. Dilute resulting solution with 10mM Tris-HCl pH7.0 and measure Abs at     260 nm. (0.7Abs)
  • Calculate the concentration by using the formulas below.
    • 0.7(Abs)×0.85×5=(2.975 OD) ⁄ 850μl= (3.5 OD) ⁄ (1000μl )
    3.5OD ⁄ 1000μl×35.1μg ⁄ OD=(122.85μg) ⁄ ml
    →0.0317×10-6 mol / ml→0.0317×106 nM

  • Concentration of AuNP (diemeter:5nm) solution ; 5.5×106 nano-particles     /liter
    •
  • Calculate the number of ahcnor oligo DNA moles we needed to prepare     AuNP-anchor oligo DNA conjugates by using the fomulas below.
    • Mol conjugated oligonucleotide=4πr2×Cn×(35pmol/cm2)×V

    r ; radius of AuNP (nm)
    Cn ; concentration of AuNP solution (nano-particles ⁄ liter)
    v ; volume of AuNP solution (L)

    ∴4π(2.5nm)2×5.5×106(particles ⁄ L)×35(pmol ⁄ cm2)×1(ml)=1.511nmol

    mol conjugated anchor oligo DNA ; 1.511nmol
    (volume of AuNP solution ; 1ml)

  • Concentration of purified anchor oligo DNA solution ; 0.317×106nM

    • 4. Mix 1ml AuNP solution and 94.64μl anchor oligo DNA solution (contains     3nmol oligo).
    5. Rotate the solution 16h at room temperature at low speed.
    6. Add 1M NaCl and 0.1M sodium phosphate buffer (137μl of each) to the     solution.
        Final concentration ; 0.1M NaCl 10mM phosphate buffer
    7. Rotate the solution 24h at room temperature at low speed.
    8. Centrifuge 60min at 45,000g.
    9. Remove the supernatant and resuspend the red oil in the same volume of     0.1M NaCl / 10mM sodium phosphate buffer.
    10. Centrifuge 60min at 45,000g.
    11. Remove the supernatant again and resuspend the red oil in the same    volume of 0.3M NaCl / 0.01% sodium azide / 10mM sodium phosphate     buffer.
    12. easure Absorbance at 520nm. (0.26Abs)
  • Calculate the concentration of the conjugate solution by using folulas     below.
    •
    Cn=(A520×1)(107×b)
    b ; path length of the cuvette (typically 1cm)
    Cn=(0.26(Abs)×1) / (107×0.5)=0.52×107M=52nM


    Method Ⅳ---Attaching AuNP on the surface of DNA origami and colum purification

    1. Mix 15μl DNA origami solution and 172.5μl AuNP-oligo solution and leave     1h at room temperature.
    2. Purify solution to remove excess staples and AuNP-oligo by using     Sephacryl S-300HR (GE healthcare). (3)
    サンプル

    Method Ⅴ---AFM observation (3)

    1. Before AFM, add 2μl glue DNA solution to 98μl purified DNA origami with     AuNP solution and leave it more than 1h.
    サンプル
    2. 1μl sample were adsorbed on the mica sheet for 5min at room     temperature and then add 50μl 1×TAE buffer.
    3. Get AFM images using Multimode 8 (Bruker).
    Table2. Sample solution for AFM
        DNA origami    
          glue DNA       
            AuNP        
      1  
    ○
    -
    -
      2  
    ○
    ○
    -
      3  
    ○
    -
    ○

    4. AFM probes ; SCANASYST-FLUID (Nitride Coated Silicon Tip on a Nitride     Cantilever), SCANASYST-FLUID＋(Silicon Tip on Nitride Lever)

    Method Ⅵ---Absorbance determination

    1. Determine absorbance at 200-900nm using V-630 UV VIS-Spectrophotometer (JASCO).
  • Cuvette ; 5μl micro cell (stepped type) (JASCO)

    • Table3. Sample solution for absorbance measurement
          DNA origami      
          glue DNA       
          AuNP      
      1  
    ○
    -
    -
      2  
    ○
    ○
    -
      3  
    ○
    -
    ○
      4  
    -
    -
    ○
      5  
    ○
    ○
    ○


    REFERENCE

    (1) Paul W.K.Rothemund. Folding DNA to create nanoscale shapes and patterns. Nature. 2006 Mar 16;440(7082):297-302. image picture
    (2) Taton TA. Preparation of gold nanoparticle-DNA conjugates. Current Protocols in Nucleic Acid Chemistry. 2002 Aug;Chapter 12:Unit 12.2.
    (3)Wickham SF, Endo M, Katsuda Y, Hidaka K, Bath J, Sugiyama H, Turberfield AJ. Direct observation of stepwise movement of a synthetic molecular transporter. Nature Nanotechnology. 2011 Mar;6(3):166-9.