Difference between revisions of "Biomod/2013/Todai/Experiment"

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   <div class="mini-title">
       <a name="Reaction_between_aptamer_embedded_in_rect_tile_and_PDGF10">2) Reaction_between_aptamer_embedded_in_rect_tile_and_PDGF</a>
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       <a name="Reaction_between_aptamer_embedded_in_rect_tile_and_PDGF10">2) Reaction between aptamer embedded in rect tile and PDGF</a>
 
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   <div class="mini-title">
 
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       <a name="Reaction_between_aptamer_(3ap-M5t10f-T0)_and_PDGF">6) Reaction_between_aptamer_(3ap-M5t10f-T0)_and_PDGF</a>
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       <a name="Reaction_between_aptamer_(3ap-M5t10f-T0)_and_PDGF">6) Reaction between aptamer (3ap-M5t10f-T0) and PDGF</a>
 
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Revision as of 23:43, 26 October 2013

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<a name="Experiment"> Experiment</a>

  • <a href="#Contents">Contents of pilot study</a>
  • <a href="#Contents">Contents of Protocols</a>
  • <a href="#PilotStudy">Pilot Study</a>
  • <a href="#Protocols">Protocols</a>
   	


  	<article>

<a name="Contents"> Contents of pilot study</a>

      	<article>
  • STEP 1: DNA strands assemble to form designed structures.
  • <a href="#STEP2">STEP 2: Subunits penetrate into the membrane.</a>
    • <a href="#hybridization_of_Cholesterol_Oligo_with_OCK">1) hybridization of cholesterol oligo with OCK</a>
    • <a href="#Preparation_of_liposome">2) Preparation of liposome</a>
    • <a href="#Flotation_assay_of_liposome_and_DNA_origami">3) Floatation assay of liposome and Rectangular tile(DNA origami)</a>

  • <a href="#STEP3">STEP 3: Subunits recognize cancer-specific proteins.</a>
  • <a href="#STEP4">STEP 4: The formed subunits oligomerize in solution.</a>
    • <a href="#Click_reaction_via_(3+2)_cycloaddition">1) Optimum time of click reaction via (3+2) cycloaddition</a>
    • <a href="#OptimumConc_SA">2) OptimumConc SA</a>
 		</article>
		</article>
		
		
		   	<article>

<a name="Contents"> Contents of protocols</a>

      	<article>
  • <a href="#STEP1">STEP 1: DNA strands assemble to form designed structures.</a>
    • <a href="#Assembling_of_OCK">1) Assembly of OCK</a>
    • <a href="#Transmission_electron_microscopy">2) Transmission electron microscopy(TEM)</a>

  • <a href="#STEP2">STEP 2: Subunits stick in the membrane.</a>
    • <a href="#Flotation_assay_[OCK]">1) Flotation assay [OCK]</a>
    • <a href="#Preparation_of_GUVs">2) Preparation of GUVs</a>
    • <a href="Preparation_of_SUVs">3) Preparation of SUVs</a>
    • <a href="#hybridization_of_Cholesterol_Oligo_with_OCK">4) hybridization of cholesterol oligo with OCK</a>

  • <a href="#STEP3">STEP 3: Recognition of target cells</a>
    • <a href="#Reaction_of_a_biotinized_oligo_to_streptavidin">1) Reaction of a biotinized oligo to streptavidin</a>
    • <a href="Reaction_between_aptamer_embedded_in_rect_tile_and_PDGF10">2) Reaction_between_aptamer_embedded_in_rect_tile_and_PDGF</a>
    • <a href="#efficient_hybridization_(changing_mixture_ratio)">3) Efficient hybridization (changing mixture ratio)</a>
    • <a href="#efficient_hybridization_(incubation_time)">4) efficient hybridization (incubation time)</a>
    • <a href="#insertion_of__hybridized_double-stranded_DNA_into_tile">5) Insertion of hybridized double-stranded DNA into tile</a>
    • <a href="#Reaction_between_aptamer_(3ap-M5t10f-T0)_and_PDGF">6) Reaction_between_aptamer_(3ap-M5t10f-T0)_and_PDGF</a>
    • <a href="Double insertion_of__hybridized_double-stranded_DNA_into_tile">7) Double insertion of hybridized double-stranded DNA into tile</a>

  • <a href="#STEP4">STEP 4: The formed subunits oligomerize in solution.</a>
    • <a href="#Oligomerization_by_streptavidin-biotin_complex">1) Oligomerization by streptavidin-biotin complex</a>
    • <a href="#Click_reaction_via_(3+2)_cycloaddition"> 2) Click reaction via (3+2) cycloaddition </a>
    • <a href="#Accelerated_Click_reaction">3) Accelerated Click reaction (using streptavidin to make the aklyne and azide reactive groups close) </a>
    • <a href="#Click_reaction_(using_hybridization_to_make_the_aklyne_and_azide_reactive_groups close)">4) Click reaction (using hybridization to make the aklyne and azide reactive groups close) </a>
    • <a href="#Click_reaction_(copper_catalyst-free)">5) Click reaction (copper catalyst-free)</a>
    • <a href="#Synthesis_of_streptavidin_mutants">6) Synthesis of streptavidin mutants</a>
 		</article>
		</article>

<a name="PilotStudy"> Pilot Study</a>

<a name="STEP2"> STEP 2: Subunits penetrate into the membrane </a>

  <article>
      <a name="hybridization_of_Cholesterol_Oligo_with_OCK">1) Hybridization of cholesterol Oligo with OCK</a>
      <figure>
        <img src="http://openwetware.org/images/d/d7/480px_OCKchol-Todai.png" width=480px height=300px >
      </figure>


[Discussion]

The result of 1 % agarose gel electrophoresis showed that the band of sample 5 and 7 were smeared, showing the successful of hybridization of cholesterol oligo with OCK (Langecker et al. (2012)). We concluded that the optimized condition for hybridization is: 1 hour incubation at room temperature with 5 times excess cholesterol oligo to OCK.

  	

</article>

<article

      <a name="Preparation_of_liposome">2) Preparation of liposome</a>
      <figure>
        <img src="http://openwetware.org/images/7/71/640px_suv_dls_popg50r60-Todai.png" width=640px height=360px >

<figcaption> The result of DLS (Viscotek, 802 DLS)

     </figcaption>
      </figure>
[Discussion]

For floating assay, uniformly-sized liposome were prepared. DLS data shows sharp peak with the mean radius of 60 nm, indicating the homogenity of liposomes.

  </article>
  
  <article>
      <a name="Flotation_assay_of_liposome_and_DNA_origami">3) Floatation assay of liposome and Rectangular tile(DNA origami)</a>
      <figure>
        <img src="http://openwetware.org/images/1/1d/640pxflotationassay-Todai.jpg" width=300px height=300px >
	<figcaption> Result of agarose gel electrophoresis of the sample of flotation assay 
The result of 1% agarose gel electrophoresis(100V,30min). In this measurement, the fluorescence of Cy5, which is

integrated into DNA origami(Rect tile[1]) ,is observed. Fraction1 is the liquid in the top layer, and fra ction 5 is in the bottom layer. Fraction 6 is the sample retrieved from precipitation. DNA origami solely was also l oaded on the extreme right lane. </figcaption>

      </figure>
      <figure>
        <img src="http://openwetware.org/images/0/0d/300pxNILGraph-Todai.PNG" width=350px height=350px >
	<figcaption> Fluorescence intensity of the samples of flotation assay(DNA Rect tile +liposome)

Although the size of liposome might change during the flotation assay(data not shown), the intensity of the fluoresc ence of NIL(Nile Red, ex 500nm, em 550~700nm ) suggests the amount of lipid membrane,liposome. The fluorescence spectrum of water was subtracted as background

     </figcaption>
      </figure>


[Discussion]

To confirm the flotation assay, mixed tiles(DNA origami) and liposomes were assayed. Five samples (fraction 1,2,..., 5, from the top) were retrieved from supermetant liquid and a sample(fraction 6) from precipitation by the addition of buffer used in assay. When the sample, tile mixed with liposomes, were assayed, tiles were observed in the top layer. The distribut ion of liposomes is observed by the fluorescence of NIL(Nile Red).

  	

</article>


<a name="STEP3"> STEP 3: Subunits recognize cancer-specific proteins</a>

<article> </article>

<a name="STEP4"> STEP 4: The formed subunits oligomerize in solution</a>

      <a name="Click_reaction_via_(3+2)_cycloaddition">
       1)Optimum time of Click reaction via (3+2) cycloaddition[4]
      </a>
  	<article>
      <figure>
        <img src="http://openwetware.org/images/a/ab/450pxclick0828-Todai.jpg" width=480px height=360px >
	<figcaption> Result of urea gel electrophoresis of the sample of click reaction
     </figcaption>
      </figure>
[Discussion]

Copper(Ⅰ) catalyzed click reaction was used to dimerize of oligo DNA(length of 20bp and 14bp) . The time cause of the reaction indicate that the click reaction is so quick(<5min).

  <article>
      <a name="OptimumConc_SA">2) Optimum concentration of SA</a>
         <figure>
        <img src="http://openwetware.org/images/1/1b/OptimumConc_SA-Todai.png" width=480px height=360px >
	<figcaption> Optimization of the mixing ratio between 
The density of the band of dimers was the highest when the mixing ratio of streptavidin to SA was 3/1, so the optimum ratio of streptavidin to OCK was 3/1.
     </figcaption>
      </figure>
  
  

</article>

  <article>
      <a name="Evaluation_of_streptavidin_mutants">3) Evaluation of streptavidin mutants</a>
         <figure>
        <img src="http://openwetware.org/images/1/1e/SA_result-Todai.png" width=400px height=400px >
      </figure>
  

We added a 6His tag to the active wild-type subunit ("alive" (A) subunit), hence no 6His tag was added to inactive subunit ("dead" (D) subunit). We mixed A and D subunits at a molar ratio of 1:1 in GuHCl and refolded in PBS. Then, refolded streptavidins were purified by Ni-NTA column and the tetramers were distinguished by non-denatured SDS-PAGE.

  

<a name="Protocols"> Protocols</a>

<a name="STEP1"> STEP 1:DNA strands assemble to form designed structures.</a>


      <a name="Assembling_of_OCK">1) Assembly of OCK[2]</a>
   
<article>


[Reagent]
     
M13mp18ss 4.5 ul
Staple mix 4.5 µL
10x OCK buffer* 1 µL
   

 *...10x OCKbuffer(f.100 ul)

Tris-HCl(ph 7.5) f.50 mM 1 M 5 µL
EDTA-Na(pH 8) f.10 mM 0.5 M 2 µL
MgCl2 f.200 mM 1 M 20 µL
NaCl f.500 mM 5 M 1 µL
MQ - - 72 µL
[Procedure]
  • mix the solutions.
  • It was annealed at 85 °C for 25 min and then at 52 °C for 3 or 4 hours.
   </article>
   


  <article>
      <a name="Transmission_electron_microscopy">
      2) Transmission electron microscopy(TEM)
      </a>
[Procedure][6]

The procedure of TEM was refered to previous researches[6].

   </article>

<a name="STEP2"> STEP 2: Subunits penetrate into the membrane.</a>


      <a name="Flotation_assay_[OCK]">1) Flotation assay [OCK]</a>
  <article>
[Reagent]
     
OCK 100 µL
Cholesterol hybridized OCK 100 µL
Liposome (1 mg/mL SUVs) 100 µL
2.25 M Sucrose buffer* 500 µL
1.6 M Sucrose buffer** 900 µL
150 mM KCl solution 100 µL
1×Flotation buffer** 600 µL
   

 *...2.25 M Sucrose buffer

HEPES-KOH (pH 7.6) 50 mM
KCl 100 mM
MgCl2 20 mM
Sucrose 2.25 M


   </table>
   

 **...1.6 M Sucrose buffer

HEPES-KOH (pH 7.6) 50 mM
KCl 100 mM
MgCl2 20 mM
Sucrose 1.6 M


   </table>
   

 ***...1×Flotation buffer

HEPES-KOH (pH 7.6) 50 mM
KCl 100 mM
MgCl2 20 mM


[Procedure]
  • Each sample was mixed as shown below:****
  •    </table>
       

     ****...Table1. Breakdown of Samples

    Sample No. 1 2 3 4
    Cholesterol hybridized OCK 50 µL 50 µL - -
    OCK - - 50 µL 50 µL
    Liposome 50 µL - 50 µL -
    150 mM aqueous KCl solution - 50 µL - 50 µL
    2.25 M Sucrose buffer 125 µL 125 µL 125 µL 125 µL
  • 225 µL of 1.6 M sucrose buffer was overlaid with 225 µL of sample mixture in centrifuge tubes (Beckman, cat#343778, 11 x 34 mm).
  • Centrifuge for 16 minutes at 100 krpm at 4 ℃ using TLA 100.2 rotor (BECKMAN COULTER) with Ultracentrifuge (BECKMAN COULTER, Optima MAX-XP).
  • 150 µL of supernatant was extracted from top to bottom for 3 times (Fraction 1 to 3) and the pellet was retrieved with 150 µL of 1×Flotation buffer (Fraction 4).
  • Fraction 1-4 of each sample were analyzed by 1 % agaraose gel electrophoresis (100V, 1 hour).
  • The Intensity of fluorescence of NileRed (Liposome) was measured with fluorescence spectrophotometer (JASCO, FP-6500) to investigate the existence of liposome in each Fraction.
  • The radiuses of liposome of each fraction were measured with DLS (Viscotek, 802 DLS).


   </article>
   
   


   


      <a name="Preparation_of_GUVs">2) Preparation of GUVs</a>
  <article>
[Reagent]
     
Lipid mix* 3 ml
150 mM KCl solution 1 µL
   

 *...Lipid mix

5 mg/mL POPC 0.1 mL
5 mg/mL POPG 0.1 mL
10 uM NileRed solution 0.13 mL
Chloroform 2.67 mL
[Procedure]
  • A lipid film was formed by evaporating 3 ml of lipid mix in a 50 ml eggplant flask, using a rotational evaporator (EYELA, model#N1110) for 10 mins.
  • The flask was kept under vacuum overnight to evaporate remaining chloroform.
  • The lipid film was resuspended in 1 mL of 150 mM KCl solution.
   </article>
   


      <a name="Preparation_of_SUVs">3) Preparation of SUVs</a>
  	<article>

Type 1: POPC 100%

[Reagent]
     
150mM KCl solution 3mL
POPC 3mg
Chloroform (99.0%) 3mL
40μM Nile Red solution 0.1mL
   
[Procedure]
  • POPC were dissolved in 3mL of Chloroform.
  • A lipid film was formed by evaporating 3mL of POPC solution in a 50mL eggplant flask, using a rotational evaporator for 5 minutes.
  • The flask was kept under vacuum overnight to evaporate remaining chloroform.
  • The lipid film was resuspended in 3mL of a 150mM KCl solution.
  • The solution was filtered through 200nm polar filter with extruder to even the size of liposome.
  • The size of liposome was measured with DLS (Viscotek 802 DLS).
  • The solution was kept at 3 degree C until usage.
   </article>
   
<article>

Type 2: POPC 50%, POPG 50%

[Reagent]
     
Lipid mix* 3 ml
150 mM KCl solution 1 µL
   

 *...Lipid mix

5 mg/mL POPC 0.1 mL
5 mg/mL POPG 0.1 mL
10 uM NileRed solution 0.13 mL
Chloroform 2.67 mL
[Procedure]
  • A lipid film was formed by evaporating 3 ml of lipid mix in a 50 ml eggplant flask, using a rotational evaporator (EYELA, model#N1110) for 10 mins.
  • The flask was kept under vacuum overnight to evaporate remaining chloroform.
  • The lipid film was resuspended in 1 mL of 150 mM KCl solution.
  • Lipid suspended solution was filtered through 100nm polar filter using extruder (Avanti) to prepare uniformly-sized liposome.
  • The size of liposome was measured with DLS (Viscotek 802 DLS).
  • The solution was kept at 3℃ until usage.
   </article>
   
  <article>
      <a name="Protocol_Hybridization_of_cholesterol_oligo_with_OCK">4) Hybridization of cholesterol oligo with OCK</a>
[Reagent]
     
OCK 48 µL
Cholesterol oligo (0.32, 0.64, 3.2, 6.4 µM) 100 µL

Marker; GeneRuler DNA Ladder Mix (Fermentas, GeneRuler DNA Ladder Mix #SM0331)

[Procedure]
  • Centrifuge for 16 minutes at 100 krpm at 4 ℃ using TLA 100.2 rotor (BECKMAN COULTER) with Ultracentrifuge (BECKMAN COULTER, Optima MAX-XP).
  • Each sample was mixed and incubated as shown below:*
  •    </table>
       

     *...Table1.

    Sample No. 1 2 3 4 5 6 7 8
    Purified OCK (40 µM) 6 µL 6 µL 6 µL 6 µL 6 µL 6 µL 6 µL 6 µL
    0.32 µM Cholesterol oligo 1.5 µL 1.5 µL - - - - - -
    0.64 µM Cholesterol oligo - - 1.5 µL 1.5 µL - - - -
    3.2 µM Cholesterol oligo - - - - 1.5 µL 1.5 µL - -
    6.4 µM Cholesterol oligo - - - - - - 1.5 µL 1.5 µL
    [Cholesterol oligo]/[OCK] (see #Note) 1/2 1/2 1 1 5 5 10 10
    Incubation time [min] 60 30 60 30 60 30 60 30
    1. Note; OCK has 4 cholesterol oligo binding sites. Therefore, we devided the molar ratio of cholesterol oligo to OCK with 4.
                   <figure>
    
            <img src="http://openwetware.org/images/6/66/OCK_Cholesterol-Todai.png" width=300px height=300px>
           
    
          </figure>
    
  • Each sample was analyzed by 1% agarose gel electrophoresis (100V, 1 hour).


   </article>
   



<a name="STEP3"> STEP 3: Subunits recognize cancer-specific proteins.</a>

<article>

  <article>
      <a name="Reaction_of_a_biotinized_oligo_to_streptavidin">1) Reaction of a biotinized oligo to streptavidin</a>
[Reagent]
     
  • materials for hybridization
  • 5ap_M-3t4e_T0 (1uM) (oligo): 5’ to 3’
  • TACTCAGGGCACTGCAAGCAATTGTGGTCCCAATGGGCTGAGTACGCGACCTCATCTTTGACCCCCAGGCAGGGAG

  • 5ap_5B_16 (10uM) (oligo, which has biotin in 5’ end):
  • Biotin-TACTCAGCCCATTGGG

  • 10x tile buffer*
  • MilliQ
  •    </table>
       

     *...10x tile buffer(f.100 µl)

    Mg(OAc)2 f.100 mM
    Tris-HCl (pH7.5) f.200 mM
    EDTA f.10 mM
  • 5ap_tile
  • M13mp18 (scaffold)
  • Cy5_Rmix (staples)
  • 10x tile buffer
  • Cy3 streptavidin (800nM)
  • [Procedure]
    • Hybridization
    • Mix materials (mentioned above in [hybridization]) in 0.2 ml PCR-tubes.
    • Incubate the mixture at the room temperature (25 ℃) for 1 hour.
    • Making 5ap_tile
    • Mix materials (mentioned above in [5ap_tile]) in 0.2 ml PCR-tubes.
    • Anneal the mixture using PCR machine (from 85 ℃ to 25 ℃, -2 ℃/min).
    • Insertion of hybridized double-stranded DNA into 5ap_tile
    • Mix hybridized double-stranded DNA and 5ap_tile in 0.2 ml PCR-tubes.
    • Incubate the mixture at 48 ℃ for 1 hour.
    • Mix the mixture and Cy3 streptavidin.
    • 1wt% Agarose-gel Electrophoresis
    • Electrophoresis the inserted 5ap_tile for 50 minutes at 100 V at 4 ℃.
    • Take photographs of the electrophoresed gel by LAS-4000.

       </article>
       
      <article>
    
          <a name="Reaction_between_aptamer_embedded_in_rect_tile_and_PDGF10">2) Reaction between aptamer embedded in rect tile and PDGF</a>
    
    [Reagent]
         
  • materials for hybridization
  • 5ap_M-3t4e_T0 (1uM) (oligo): 5’ to 3’
  • TACTCAGGGCACTGCAAGCAATTGTGGTCCCAATGGGCTGAGTACGCGACCTCATCTTTGACCCCCAGGCAGGGAG

  • 5ap_M-3t4e_T-3(1µM) (oligo):
  • TACTCAGGGCACTGCAAGCAATTGTGGTCCCAATGGGCTGAGTAGACCTCATCTTTGACCCCCAGGCAGGGAG

  • 5ap_M-3t4e_T-1(1µM) (oligo):
  • TACTCAGGGCACTGCAAGCAATTGTGGTCCCAATGGGCTGAGTAGCGACCTCATCTTTGACCCCCAGGCAGGGAG

  • 5ap_M-3t4e_T7(1µM) (oligo):
  • TACTCAGGGCACTGCAAGCAATTGTGGTCCCAATGGGCTGAGTAtttttttCGCGACCTCATCTTTGACCCCCAGGCAGGGAG

  • 5ap_5B_16 (10uM) (oligo, which has biotin in 5’ end):
  • Biotin-TACTCAGCCCATTGGG

  • 10x tile buffer*
  • MilliQ
  •    </table>
       

     *...10x tile buffer(f.100 µl)

    Mg(OAc)2 f.100 mM
    Tris-HCl (pH7.5) f.200 mM
    EDTA f.10 mM
  • 5ap_tile
  • M13mp18 (scaffold)
  • 5ap_Rmix (staples)
  • 10x tile buffer
  • PDGF(dye 45nM)
  • [Procedure]
    • Hybridization
    • Mix materials (mentioned above in [hybridization]) in 0.2 ml PCR-tubes.
    • Incubate the mixture at the room temperature (25 ℃) for 1 hour.
    • Making 5ap_tile
    • Mix materials (mentioned above in [5ap_tile]) in 0.2 ml PCR-tubes.
    • Anneal the mixture using PCR machine (from 85 ℃ to 25 ℃, -2 ℃/min).
    • Insertion of hybridized double-stranded DNA into 5ap_tile
    • Mix hybridized double-stranded DNA and 5ap_tile in 0.2 ml PCR-tubes.
    • Incubate the mixture at 48 ℃ for 1 hour.
    • Mix the mixture and PDGF.
    • Incubate at 37 ℃ for 30 min and then at 4 ℃ for 25 hours.
    • 1wt% Agarose-gel Electrophoresis
    • Electrophoresis the inserted 5ap_tile for 50 minutes at 100 V at 4 ℃.
    • Stain the gel by SYBR Gold in TBE.
    • Take photographs of the electrophoresed gel by LAS-4000.

       </article>
       


      <article>
    
          <a name="efficient_hybridization_(changing_mixture_ratio)">3) Efficient hybridization (changing mixture ratio)</a>
    
    [Reagent]
         
  • 5ap_M-3t4e_T0 (1 µM): 5’ to 3’
  • TACTCAGGGCACTGCAAGCAATTGTGGTCCCAATGGGCTGAGTACGCGACCTCATCTTTGACCCCCAGGCAGGGAG

  • 5ap_5B_16 (1 or 10 µM): Biotin in 5’ end
  • Biotin - TACTCAGCCCATTGGG

  • 10x tile buffer*
  • MilliQ
  •    </table>
       

     *...10x tile buffer

    Mg(OAc)2 f.100 mM
    Tris-HCl (pH7.5) f.200 mM
    EDTA f.10 mM


    [Procedure]
    • Mix materials to make samples, following the ratio written in Table.3.*
    •    </table>
         

       *...Table.3

      Sample No. 1 2 3 4 5 6 7 8
      5ap_M-3t4e_T0 (1 µM) 3 µL - - 3 µL 3 µL 3 µL 3 µL 3 µL
      5ap_5B_16 (10 µM) - - - - - 1.5 µL 3 µL 6 µL
      5ap_5B_16 (1 µM) - 3 µL - 3 µL 6 µL - - -
      10x tile buffer 1 µL 1 µL 1 µL 1 µL 1 µL 1 µL 1 µL 1 µL
      MilliQ 6 µL 6 µL 9 µL 3 µL - 4.5 µL 3 µL -
      Ratio of concentration of 5ap_5B_16 to 5ap_M-3t4e_T0 - - - 1 % 2 % 5 % 10 % 20 %
    • Apply the samples to 10 % Native-PAGE for 85 minutes at 100 V at 4 ℃.
    • Stain the gel by SYBR Gold in TBE.
    • Take a photograph of the gel by LAS-4000.
       </article>
       
    <article>
          <a name="efficient_hybridization_(incubation_time)">4) efficient hybridization (incubation time)</a>
    
    [Reagent]
         
  • 5ap_M-3t4e_T0 (1 µM): 5’ to 3’
  • TACTCAGGGCACTGCAAGCAATTGTGGTCCCAATGGGCTGAGTACGCGACCTCATCTTTGACCCCCAGGCAGGGAG

  • 5ap_5B_16 (10 µM): (biotin in 5’ end)
  • Biotin - TACTCAGCCCATTGGG

  • 10x tile buffer*
  • MilliQ
  •    </table>
       

     *...10x tile buffer(f.100 µl)

    Mg(OAc)2 f.100 mM
    Tris-HCl (pH7.5) f.200 mM
    EDTA f.10 mM


    [Procedure]
    • Mix the materials in 0.2 ml PCR-tubes.
    • Denature the oligos at 95 ℃ for 30 seconds.
    • Incubate the mixture at room temperature (25 ℃).
    • Freeze the samples into nitrogen liquid at planned incubation time.
    • Pick up the samples out from nitrogen liquid immediately before applying into gel.
    • Apply the samples to 10 % Native PAGE for 85 minutes at 100 V at 4 ℃.
    • Take a photograph of the electrophoresed gel by LAS-4000.

       </article>
       
    <article>
          <a name="insertion_of__hybridized_double-stranded_DNA_into_tile">5) Insertion of hybridized double-stranded DNA into tile</a>
    
    [Reagent]
         

    hybridization

  • 5ap_M-3t4e_T0 (1 µM) (oligo): 5’ to 3’
  • TACTCAGGGCACTGCAAGCAATTGTGGTCCCAATGGGCTGAGTACGCGACCTCATCTTTGACCCCCAGGCAGGGAG

  • 5ap_5B3G_16 (1 µM) (oligo, which has biotin in 5’ end and Cy3 in 3’ end):
  • Biotin–TACTCAGCCCATTGGG–Cy3

  • 10x tile buffer*
  • MilliQ
  •    </table>
       

     *...10x tile buffer(f.100 µl)

    Mg(OAc)2 f.100 mM
    Tris-HCl (pH7.5) f.200 mM
    EDTA f.10 mM

    5ap_tile

  • M13mp18 (scaffold)
  • 5ap_Rmix (staples)
  • 10x tile buffer
  • [Procedure]
      Hybridization
    • Mix materials (mentioned above in [hybridization]) in 0.2 ml PCR-tubes.
    • Incubating the mixture at the room temperature (25 ℃) for 1 hour.
    • Making 5ap_tile

    • Mix materials (mentioned above in [5ap_tile]) in 0.2 ml PCR-tubes.
    • Anneal the mixture using PCR machine (from 85 ℃ to 25 ℃, -2 ℃/min).
    • Insertion of hybridized double-stranded DNA into 5ap_tile

    • Mix hybridized double-stranded DNA and 5ap_tile in 0.2 ml PCR-tubes.
    • Incubate the mixture at 48 ℃ for 1 hour.
    • 1 wt% Agarose-gel Electrophoresis

    • Electrophoresis the inserted 5ap_tile for 50 minutes at 100 V at 4 ℃.
    • Take photographs of the electrophoresed gel by LAS-4000.
       </article>
       
    <article>
          <a name="Reaction_between_aptamer_(3ap-M5t10f-T0)_and_PDGF">6) Reaction between aptamer (3ap-M5t10f-T0) and PDGF</a>
    
    [Reagent]
         
  • materials for the reaction
  • 10x tile buffer f. 1x tile buffer
    1 µM aptamer f. 0.3µM
    1 µM Ladder151515_1 f. 0.3µM
    PDGF (dye 45nM) f. 20mM
  • materials for the electrophoresis
  •    <gel for 10% Native-PAGE>
    
    MilliQ 7.9 mL
    30% Acrylamide mix 6.7 mL
    1.5 M Tris-HCl (pH 8.8) 5 mL
    1 M MgCl2 200 µL
    10 % APS 80 µL
    TEMED 80 µL
       <Electrophoresis buffer for 10 % Native-PAGE>
         1x TBE
    
       <materials for stain>
    
    Electrophoresis buffer for 10 % Native-PAGE 50 mL
    SYBR Gold 5 µL
       <others>
    
    Loading buffer 20 % glycerol (as used 6x)
    Marker Cy5 38 mer


    [Procedure]
    • Mix the solutions as shown below:*
    •    </table>
         

       *...Table.2

      Sample No. 1 2 3 4 5 6
      10x tile buffer 1 µL 1 µL 1 µL 1 µL 1 µL 1 µL
      1 µM aptamer 3 µL - - 3 µL - -
      1 µM Ladder151515_1 - - - - 3 µL 3 µL
      PDGF (dye 45 nM) - 6 µL - 6 µL - 6 µL
      MilliQ 6 µL 3 µL 9 µL - 6 µL -
    • Incubate at 37 ℃ for 30 min and then at 4 ℃ for 30 min.
    • Make 10 % Native PAGE gel as mentioned above.
    • Add loading buffer into each samples.
    • Apply the samples to 10 % Native-PAGE for 85 minutes at 100V at 4℃.
    • Take a photograph of the electrophoresed gel by LAS-4000 for Cy5.
    • Stain the gel by SYBR Gold for 20 min.
    • Take a photograph by LAS-4000.
       </article>
       


    </article>

      <article>
    
          <a name="Double insertion_of__hybridized_double-stranded_DNA_into_tile">7) Double insertion of hybridized double-stranded DNA into tile</a>
    
    [Reagent]
         

    hybridization

  • 5ap_R125_T-3_Cy5 (1 µM) (oligo):
  • TACTCAGGGCACTGCAAGCAATTGTGGTCCCAATGGGCTGAGTAGACCTCATCTTTGACCCCCAGGCAGGGAG

  • 5ap_5B3T30_16_Cy3 (1 µM) (oligo, which has Cy3 in 5’ end):
  • TACTCAGCCCATTGGGttttttttttttttttttttttttttttttAAAACACTGCTCCATGTTACTTAACAAAGCT

  • 10x tile buffer*
  • MilliQ
  •    </table>
       

     *...10x tile buffer(f.100 µl)

    Mg(OAc)2 f.100 mM
    Tris-HCl (pH7.5) f.200 mM
    EDTA f.10 mM

    5ap_tile

  • M13mp18 (scaffold)
  • 5ap_Rmix (staples)
  • 10x tile buffer
  • [Procedure]
      Hybridization
    • Mix materials (mentioned above in [hybridization]) in 0.2 ml PCR-tubes.
    • Incubating the mixture at the room temperature (25 ℃) for 1 hour.
    • Making 5ap_tile

    • Mix materials (mentioned above in [5ap_tile]) in 0.2 ml PCR-tubes.
    • Anneal the mixture using PCR machine (from 85 ℃ to 25 ℃, -2 ℃/min).
    • Insertion of hybridized double-stranded DNA into 5ap_tile

    • Mix hybridized double-stranded DNA and 5ap_tile in 0.2 ml PCR-tubes.
    • Incubate the mixture at 48 ℃, 46℃, 44℃, 42℃,or 40℃ for 1 hour.
    • 1 wt% Agarose-gel Electrophoresis

    • Electrophoresis the inserted 5ap_tile for 50 minutes at 100 V at 4 ℃.
    • Stain the gel by SYBR Gold in TBE.
    • Take photographs of the electrophoresed gel by LAS-4000.
       </article>
       


    <a name="STEP4"> STEP 4: The formed subunits oligomerize in solution.</a>

      <article>
    
          <a name="Dimerization_of_OCK--using_biotin,_streptavidin_and_click_ reaction">1) Dimerization of OCK--using biotin, 	streptavidin and click reaction</a>
    
    [Reagent]
         
    OCK (90 nM) 8 µL
    Streptavidin (190 nM) 2 µL
    CuSO4 aq (8 mM) 1 µL
    THTA (32.5 mM) 1 µL
    Sodium ascorbate (3.25 mM) 1 µL
       


    [Procedure]
    • 7.4 µL of OCK and 1 µL Streptavidin (190 nM) were mixed and kept at room temperature (27 ℃) for an hour. (Mix1)
    • 10 µL of Mix1 and 1 µL of Sodium ascorbate (3.25 mM) were mixed and then 1 µL of CuSO4 aq (8 mM) was added into that solution.
    • The solution was mixed and 1µL of THTA (20 mM) was added in it and mixed.
    • That solution was kept at room temperature (27 ℃) for a day.
       </article>
       


          <a name="Click_reaction_via_(3+2)_cycloaddition">
          2) Click reaction via (3+2) cycloaddition
          </a>
    
      <article>
    
    [Reagent]
         
    azide solution (10μM) 3μL
    alkyne solution (10μM) 3μL
    CuSO4 solution (50mM) 1μL
    THTA solution (100mM) 1μL
    sodium ascorbate solution (100mM) 1μL
       
    [Procedure][4]
    • The above all solutions were mixed, using a vortex.
    • The solution was kept at room temperature.
       </article>
       
    <article>
          <a name="Accelerated_Click_reaction">3) Accelerated Click reaction (using streptavidin to make the aklyne and azide 	reactive groups close) </a>
    
    [Reagent]
         
    2x barrel buffer 6 µL
    alkyne oligo (carrying biotin) (15 µM) 1 µL
    azide oligo (carrying biotin) (15 µM) 1 µL
    streptavidin (500 µM) 1 µL
       


    [Procedure]
    • mix reagents
    • incubate the tube at 37 ℃ for indicated reaction time.
    • boil at 95 ℃ for 30 minutes to break down streptavidin
       </article>
       
    <article>
          <a name="Click_reaction_(using_hybridization_to_make_the_aklyne_and_azide_reactive_groups close)">4) Click reaction 	(using hybridization to make the aklyne and azide reactive 	groups close) </a>
    
    [Reagent]
         
    2x barrel buffer 7 µL
    alkyne oligo (15 µM) 1 µL
    azide oligo (15 µM) 1 µL
    scaffold (15 µM) 1 µL
       


    [Procedure]
    • mix reagents
    • incubate the tube at 37 ℃.
    • add loading buffer into the reaction mixture and boil at 95 ℃ for 5 minutes to denature the double strand to single strand.
          <a name="Click_reaction_(copper_catalyst-free)">5) Click reaction (copper catalyst-free)</a>
    
    [Reagent]
         
    2x barrel buffer 7 µL
    alkyne oligo (15 µM) 1 µL
    azide oligo (15 µM) 1 µL
    scaffold (15 µM) 1 µL
       
    [※※ 2x barrel buffer]
          
    1M Tris (pH 7.5) 5 µL
    0.5M EDTA 2 µL
    5M NaCl 1 µL
    MQ 32 µL
       
    [Procedure]
    • mix reagents
    • incubate the tube at 37 ℃.
    • add loading buffer into the reaction mixture and boil at 95 ℃ for 5 minutes to denature the double strand to single strand.
       </article>
    


    <article>
    
          <a name="Synthesis_of_streptavidin_mutants">
          6) Synthesis of streptavidin mutants
          </a>
    


    [Procedure][7],[8]

    Mono-, di-, tri-, tetra-valent streptavidin were prepared as described [7,8] with some modifications. Shortly, BL21 Star (DE3) pLysSRARE and C43 (DE3) was transformed with pET21a(+) SA-Alive-his or pET21a(+) SA-Dead plasmids and cultured in LB at 37℃. Collected cells were resuspended in B-PER (Pierce) and inclusion bodies were purified, and dissolved in 6M guanidinium hydrochloride (GuHCl; pH 1.5). After mixing the unfolded subunits in desired ratio, the unfolded subunits were refolded by rapid dilution into PBS, then concentrated by ammonium sulfate precipitation. After dialyzed 3 x against PBS, refolded streptavidin were purified by Ni-NTA column (GE 17-5248-02) using AKTA system (GE AKTAexplorer 10S). Fractionized samples were concentrated by Amicon Ultra (Millipore).

       </article>
    


    <a name="Reference"> Reference</a>

           <a name="proref-1">
           [1] Folding DNA to create nanoscale shapes and patterns
           </a>
    
              Rothemund, P. W.
    
                 Nature 440, 297–302 (2006)
    
           <a name="proref-1">
           [2] Rapid Folding of DNA into Nanoscale Shapes at Constant Temperature
           </a>
    
               Jean-Philippe J. Sobczak, Thomas G. Martin, Thomas Gerling, Hendrik Dietz
    
                 Science, 2012, 338, 1458
    
           <a name="proref-1">
           [3] Transcription Regulation System Mediated by Mechanical Operation of a DNA      &nbs	p;Nanostructure
           </a>
    
              Masayuki Endo, Ryoji Miyazaki, Tomoko Emura, Kumi Hidaka, and Hiroshi Sugiyama
    
                 Journal of the American Chemical Society, 2012, 134, 2852-2855
    
           <a name="proref-1">
           [4] the protocol of Jena Bioscience GmbH
           </a>
    
              <a target="_blank" href="http://www.jenabioscience.com" style="color:#e00000">
              http://www.jenabioscience.com</a>
    
           <a name="proref-1">
           [5] Substrate-Assisted Assembly of Interconnected Single-Duplex DNA Nanostructures
           </a>
    
              Shogo Hamada, Satoshi Murata Prof.
    
                 Angewandte Chemie International Edition,2009,48(37),6820–6823
    
           <a name="proref-1">
           [6] A primer to scaffolded DNA origami.</a>
    
              Castro CE, Kilchherr F, Kim DN, Shiao EL, Wauer T, Wortmann P, Bathe M
    

    and Dietz H.

                 Nat Methods 221-229 (2011, Mar;8(3))
    
           <a name="proref-1">
           [7] A monovalent streptavidin with a single femtomolar biotin binding site.</a>
    
              Howarth M, Chinnapen DJ, Gerrow K, Dorrestein PC, Grandy MR, Kelleher NL, El-Husseini A and Ting AY.
    
                 Nat Methods 267-273 (2006, Apr;3(4))
    
           <a name="proref-1">
           [8] Imaging proteins in live mammalian cells with biotin ligase and monovalent streptavidin.</a>
    
             Howarth M and Ting AY.
    
                 Nat Protoc 534-545 (2008, Mar;3(3)); doi: 10.1038/nprot.2008.20.
    
      







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