Biomod/2014/Kashiwa/Protocol

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<body> PROTOCOL



<a href="#" onclick="HideCBox('CBoxBody1'); return false;" title="折りたたみ／復元">[show/hide]</a>

  Contents：

  • <a href=#1> 1. The Sensing System: The Receptor </a>
    • <a href="#1-1">1-1. Preparing the components </a>
      • <a href="#1-1-1">1-1(a). Folding the Wall </a>
      • <a href="#1-1-2">1-1(b). Producing MISTIC </a>
      • <a href="#1-1-3">1-1(c). Designing the Activator </a>
    • <a href="#1-2">1-2. Embedding the Wall into the liposome </a>
    • <a href="#1-3">1-3. Linking the Activator to the liposome </a>
    • <a href="#1-4">1-4. Combining the Wall and the Activator </a>
    • <a href="#1-5">1-5. Separating the Wall from the Activator </a>
  • <a href="#2">2. The Moving System: The Motor </a>
    • <a href="#2-1">2-1. Producing the Motor-Monomer </a>
      • <a href="#2-1-1"> 2-1(a). Folding the Motor-Monomer body </a>
      • <a href="#2-1-2"> 2-1(b). Synthesizing divalent SA </a>
      • <a href="#2-1-3"> 2-1(c). Equipping the Motor-Monomer with divalent SA </a>
    • <a href="#2-2">2-2. Deactivating and reactivating the binding capacity of streptavidin </a>
    • <a href="#2-3">2-3. Putting the Motor-Monomers into the liposome </a>


<a name="1-1"> </a>
<a name="1-1-1"> </a>

<a name="contents"> 1. Developing the sensing system</a>

1-1. The Receptor by itself

1-1-1. Design of the Receptor hetero-units

• Make the hetero-units

<tbody> </tbody>
Reagents (f. 40.0 µL)
M13mp18ss 18.0 µL
Staple mix 18.0 µL
10 × Receptor buffer¹ 4.0 µL


¹10 × Receptor buffer — 50 mM Tris (HCl pH 7.5), 10 mM EDTA-Na (pH 8),
100 mM MgCl2, 500 mM NaCl

<a name="1-1-2"> </a>

Procedure

1) The solution was mixed.

2) The mixture was annealed at 47.5 °C for 4 hours.

3) The mixture was purified by spin column.

4) The mixture was analyzed by 1% agarose gel electrophoresis (100V, 40 min).



1-1-2. Dimerization mechanism of the Receptor

<tbody> </tbody>
Reagents (f. 20.0 µL)
10 µM Aptamers 3.5 µL
1 µM Alpha - thrombin 3.5 µL
10 × physiological buffer¹ 2.0 µ L
MQ 11.0 µ L

¹10 × physiological buffer — 200 mM Tris - HCl (pH 7.5), 1.4 M NaCl, 50 mM KCl,
10 mM CaCl 2, 10 mM MgCl 2, 5% (v/v) Glycerol

<a name="1-1-3"> </a>

Procedure

1) Aptamer solution was mixed.

2) The solution was annealed from 90 °C to 24 °C, decrease by 0.4 °C / min.

3) Thrombin-solution was added to the solution and incubated at room temperature for 1 hour.

4) The mixture was analyzed with Native-PAGE (200 V 25 min, 250 V 40 min).

5) The gel was stained by EtBr for 30 minutes.

6) The gel was observed with UV and fluorescence by LAS - 4000.



1-1-3. Emission of the Initiator

<tbody> </tbody>
Reagents (f. 20.0 µL 25 nM)
100 nM Biotin modified single strand J’ 5.0 µL
100 nM The Mismatch modified Strand J 5.0 µL
100 nM The Initiator 5.0 µL
Cy5-modified Streptavidin (SA) 5.0 µL


Procedure

1) Strand J and Initiator were mixed and annealed at 85 °C,67 °C,50 °C,25 °C for 5 minutes each (mismatch factor 20 %).

2) Biotin modified single strand and SA were mixed and incubated at room temperature for 15 minutes.

3) The reagents were mixed and incubated at room temperature for 1 hour.

4) The mixture was analyzed with Native - PAGE (200 V 40 min, 250 V 40 min).

5) The gel was stained by EtBr for 30 minutes

6) The gel was observed with UV and fluorescence by LAS - 4000.

<a name="1-2"> </a>
<a name="1-2-1"> </a>

1-2. The Receptor on the liposome

1-2-1. Penetration of the Receptor hetero-units to the liposome

Ⅰ. Preparation of GUVs

GUV s were made as shown in <a href=“#2-2-1”>”Putting the Monomer into a liposome".</a>


Ⅱ. Preparation of LUVs

<tbody> </tbody>
Reagents (f. 1280 µL)
10 mg/mL POPC 200.0 µL
25 mg/mL POPG 80.0 µL
150 mM KCl solution 1.0 mL

Procedure

1) A lipid film was formed by evaporating POPC and POPG in a tube.

2) The tube was kept under vacuum overnight to evaporate remaining chloroform.

3) The lipid film was resuspended in 1 mL of 150 mM KCl solution.
Temperature was controlled to be 40 °C during suspension.

4) The solution was kept at 4 °C or -80°C and sample was sonicated before usage.



Ⅲ. Penetration of the Receptor into liposomes

<tbody> </tbody>
Reagents (f. 100.0 µL)
Liposomes (LUVs , GUVs) 50.0 µL
Cholesterol hybridized Receptor¹ 50.0 µL

¹Cholesterol hybridized Receptor — x µM Purified Receptor, 160 x µM Cholesterol oligomer


Procedure

1)Purified Receptor and cholesterol oligomer were mixed and incubated at room temperature for 60 minutes.

2)The reagents were mixed and incubated at room temperature for 30 minutes.



Ⅳ. Flotation assay

<tbody> </tbody>
Reagents (f. 2.4 mL)
The Receptor 100.0 µL
Cholesterol hybridized Receptor 100.0 µL
Liposomes 100.0 µL
2.25 M Sucrose buffer¹ 500.0 µL
1.6 M Sucrose buffer¹ 900.0 µL
150 mM KCl solution 100.0 µL
1 × Flotation buffer² 600.0 µL

¹1.6, 2.25 M Sucrose buffer — 50 mM HEPES - KOH (pH 7.6), 100 mM KCl, 20 mM MgCl2, 1.6, 2.25 M Sucrose

²1 × Flotation buffer — 50 mM HEPES - KOH (pH 7.6), 100 mM KCl, 20 mM MgCl2

Procedure

1) Each sample was mixed as shown below　<a href="#table1">(table1)</a>.

2) 1.6 M Sucrose buffer was overlaid with sample mixture in centrifuge tubes (Beckman, cat#343778, 11 × 34 mm).

3) Each sample was centrifuged for 16 minutes at 100 krpm at 4 °C using TLA 100.2 rotor (BECKMAN COULTER) with Ultracentrifuge (BECKMAN COULTER, Optima MAX-XP).

4) Supernatant (each 150 µL) was extracted from top to bottom for 3 times (Fraction 1 to 3) and the pellet was retrieved with 1 × Flotation buffer (Fraction 4).

5) Fraction 1-4 of each sample were analyzed by 1 % agaraose gel electrophoresis (100V, 1 hour).

<a name="table1"></a>

6)10 µM Nile Red was added to each fraction of sample 1 and 3. (30 µL) (6 µL) The Intensity of fluorescence of NileRed (liposomes) was measured with fluorescence spectrophotometer (JASCO, FP-6500) to investigate the existence of liposomes in each Fraction.


<tbody> </tbody>
Table 1. Breakdown of Samples
sample No. 1 2 3 4
Cholesterol hybridized Receptor 50.0 µL 50.0 µL — —
Receptor — — 50.0 µL 50.0 µL
Liposome 50.0 µL — 50.0 µL —
150 mM aqueous KCl solution — 50.0 µL — 50.0 µL
2.25 M Sucrose buffer 125.0 µL 125.0 µL 125.0 µL 125.0 µL

<a name="1-2-2"> </a> <a name="1-2-3"> </a>


<a name="1-2-2"> </a>

1-2-2. Dimerization mechanism of the Receptor on the liposome

1-2-3. Emission of the initiator in the liposome


<a name="2"> </a>
<a name="2-1"> </a>
<a name="2-1-1"> </a>

<a name="contents"> 2. Developing the moving system</a>

2-1. The Motor by itself

2-1-1. Design of the Motor-Monomer

• Make the Moter-Monomer

<tbody> </tbody>
Reagents (f. 60.0 µL)
Staple mix 17.5 µL
M 13 mp 18 ss 18.3 µL
TE¹ 18.2 µL
10 × tile buffer² 6.0 µL

¹TE — 10 mM Tris - HCl (pH 8.0), 1 mM EDTA

²10 × tile buffer — 100 mM Mg (OAc) 2, 200 mM Tris-HCl (pH 7.5), .10 mM EDTA

<a name="2-1-2"> </a>

Procedure

1) The reagents were mixed.

2) The mixture was annealed at 55 °C for 3 hours.

3) The mixture was analyzed by 1 % agarose gel electrophoresis.

4) The gel was stained by EtBr for 30 minutes.

5) A photo of the gel was taken by LAS - 4000.



2-1-2. Formation of the simple Polymer

<tbody> </tbody>
Reagents (f. 20.0 µL)
Purified Monomer X 10.0 µL
Purified Monomer Y 10.0 µL

<a name="2-1-3"> </a>

Procedure

1) Monomers were made as shown in “Making DNA origami monomers”.

2) Each Monomer solution was purified by spin column.

3) The reagents were mixed.

4) The mixture was incubated at room temperature for 24 hours.

5) The mixture was analyzed by 1 % agarose gel electrophoresis (100 V, 80 min).

6) The gel was stained by EtBr for 30 minutes.

7) The gel was taken a photo by LAS - 4000.




<a name="2-2"> </a>

2-1-3. Controlling the ring - opening polymerization

<a name="2-2-1"> </a>

2-2. The Motor in the liposome

2-2-1. Putting the Motor - Monomers into the liposome

<tbody> </tbody>
Reagents (f.1420 µL)
Lipid mix ¹ 400.0 µL
Glucose 500.0 µL
Emulsion mix² 520.0 µL

¹3 mM Lipid mix — POPC, Paraffin

²Emulsion mix — 0.5 mM Lipid mix, 50 mM HEPES, 10 mM EDTA,
400 mM Sucrose,Pyranine, The Motor-Monomers

<a name="2-2-2"> </a>

Procedure

1) Glucose (500 µL) was taken into a tube as outer solution.

2) Lipid mix (0.5 mM) was added on the glucose solution carefully.

3) Emulsion mix was added on the top and rapidly centrifuged at 4 °C, 1800 rpm for 10 minutes.

4) Centrifuged again at 4 °C, 5000 rpm for 10 minutes.

5) The upper layer was removed and GUVs were collected by micropipette.

6) The GUVs were stained by Nile red and observed with a confocal microscope.




2-2-2. Formation of the simple Polymer in the liposome

2-2-3. Controlling the ring opening polymerization in the liposome




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