Biomod/2014/Kashiwa/Protocol

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

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

  Contents：

  • <a href=#1>1. Developing the sensing system </a>
    • <a href="#1-1">1-1. The Receptor by itself </a>
      • <a href="#1-1-1">1-1-1. Design of the Receptor hetero - units </a>
      • <a href="#1-1-2">1-1-2. Dimerization mechanism of the Receptor </a>
      • <a href="#1-1-3">1-1-3. Emission of the initiator </a>
    • <a href="#1-2">1-2. The Receptor on the loposome </a>
      • <a href="#1-2-1">1-2-1. Penetration of the Receptor hetero - units to the liposome </a>
      • <a href="#1-2-2">1-2-2. Dimerization mechanism of the Receptor on the liposome </a>
      • <a href="#1-2-3">1-2-3. Emission of the initiator in the liposome </a>
  • <a href="#2">2. Developing the moving system </a>
    • <a href="#2-1">2-1. The Motor by itself </a>
      • <a href="#2-1-1">2-1-1. Design of the Motor - Monomer </a>
      • <a href="#2-1-2">2-1-2. Formation of the simple Polymer </a>
      • <a href="#2-1-3">2-1-3. Controlling the ring - opening polymerization </a>
    • <a href="#2-2">2-2. The Motor in the liposome </a>
      • <a href="#2-2-1">2-2-1. Putting the Motor - Monomers into the liposome </a>
      • <a href="#2-2-2">2-2-2. Formation of the simple Polymmer in the liposome </a>
      • <a href="#2-2-3">2-2-3. Controlling the ring opening polymerization in the liposome </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 units

<tbody> </tbody>
Reagents (f. 40 µL)
M13mp18ss 18 µL
Staple mix 18 µL
10 × Receptor buffer¹ 18 µL


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

Procedure

1) The solutions were 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).



<a name="1-1-2"> </a>



1-1-2. Dimerization mechanism of the Receptor

<tbody> </tbody>
Reagents (f. 20 µL)
Aptamers (f. ????? µM) ?????
Alpha - thrombin (f. 200 nM) ?????
10 × physiological buffer&suo1; ?????
<tbody> </tbody>
¹10 × physiological buffer(f. 10mL)
1 M Tris - HCl (pH 7.5) (f. ?????) 2 mL
3 M NaCl (f. ?????) 4.67 mL
1 M KCl(f.?????) 500 µL
100 mM CaCl 2 (f. ?????) 1 mL
1 M MgCl 2 (f. ?????) 100 µL
MQ 1.23 mL
100% Glycerol (f. ??????) 0.5 mL

Procedure

1) Aptamer solutions were mixed.

2) The solutions were annealed from 90 °C to 24 °C, decrease by 0.4 °C / min

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

4) The mixture was analyzed with Native-PAGE (??? V , ?? 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="1-1-3"> </a>



1-1-3. Emission of the initiator

<tbody> </tbody>
Reagents (f. 20 µL)
100 mM Biotin modified single strand 5 µL
100 mM Strand A 5 µL
Initiator 5 µL
SA (Cy 5) 5 µL

Procedure

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

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 ○○ hours.

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>



1-2. The Receptor on the loposome

<a name="1-2-1"> </a>



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

Ⅰ. Preparation of GUVｓ

GUVs were made as shown in "Putting the Monomer into a liposome".


Ⅱ. Preparation of LUVｓ

<tbody> </tbody>
Reagents
50 mM HEPES 500 µL
DTT (f. 1 mM) ????? µL
POPC 20 mg

COMING SOON!



Ⅲ. Hybridization of cholesterol oligomer with the Receptor

<tbody> </tbody>
Reagents (f. 7.5 µL)
Purified Receptor (0.05 µM) 6 µL
Cholesterol oligomer (8 µM) 1.5 µL

Procedure

The reagents were mixed and incubated at room temperature for 60 minutes.


Ⅳ. Penetration of the Receptor into liposomes

<tbody> </tbody>
Reagents
Liposomes (LUVs , GUVs) 50 µL
Cholesterol hybridized Receptor 50 µL

Procedure

The reagents were mixed and incubated at room temperature for 15 minutes.


Ⅴ. Flotation assay

<tbody> </tbody>
Reagents
The Receptor 100 µL
Cholesterol hybridized Receptor 100 µL
Liposomes (? mg/mL LUVs) 100 µL
2.25 M Sucrose buffer¹ 500 µL
1.6 M Sucrose buffer² 900 µL
150 mM KCl solution 100 µL
1 × Flotation buffer³ 600 µL
<tbody> </tbody>
¹2.25 M Sucrose buffer (f. ????)
HEPES - KOH (pH 7.6) (f. 50 mM) ?????
KCl (f. 100 mM) ?????
MgCl2 (f. 20 mM) ?????
Sucrose (f. 2.25 M) ?????
<tbody> </tbody>
²1.6 M Sucrose buffer (f. ????)
HEPES - KOH (pH 7.6) (f. 50 mM) ?????
KCl (f. 100 mM) ?????
MgCl2 (f. 20 mM) ?????
Sucrose (f. 1.6 M) ?????
<tbody> </tbody>
³1 × Flotation buffer (f. ?????)
HEPES - KOH (pH 7.6) (f. 50 mM) ?????
KCl (f. 100 mM) ?????
MgCl2 (f. 20 mM) ?????


Procedure

1) Each sample was mixed as shown below　(table1).

2) 225 µL of 1.6 M sucrose buffer was overlaid with 225 µL of sample mixture in centrifuge tubes (Beckman, cat#343778, 11 × 34 mm).

3) Centrifuge for 16 minutes at 100 krpm at 4 ℃ using TLA 100.2 rotor (BECKMAN COULTER) with Ultracentrifuge (BECKMAN COULTER, Optima MAX-XP).

4) 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).

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

6) The Intensity of fluorescence of NileRed (Liposomes) was measured with fluorescence spectrophotometer (JASCO, FP-6500) to investigate the existence of liposomes in each Fraction.

7) The radiuses of liposome of each fraction were measured with DLS (Viscotek, 802 DLS).

<tbody> </tbody>
Table 1. Breakdown of Samples
sample No. 1 2 3 4
Cholesterol hybridized Receptor 50 µL 50 µL — —
Receptor — — 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



<a name="1-2-2"> </a>



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

<a name="1-2-3"> </a>



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


<a name="2"> </a>



2. Developing the moving system

<a name="2-1"> </a>



2-1. The Motor by itself

<a name="2-1-1"> </a>



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

モノマーの形成

<tbody> </tbody>
Reagents (f. 60 µL)
Staple mix 17.5 µL
M 13 mp 18 ss 18.3 µL
TE¹ 18.2 µL
10 × tile buffer² 6.0 µL
<tbody> </tbody>
¹TE (f. 50 mL)
1 M Tris - HCl (pH 8.0) (f. 10 mM) 0.5 mL
0.5 M EDTA (f. 1 mM) 0.1 mL
MQ 49.4 mL
<tbody> </tbody>
²10 × tile buffer(f. 500 mL)
Mg (OAc) 2 (f. 100 mM) 10.73 g
1.0 M Tris-HCl (pH 7.5) (f. 200 mM) 100 mL
0.5 M EDTA (f. 10 mM) 10 mL
MQ up to 500 mL

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



<a name="2-1-2"> </a>



2-1-2. Formation of the simple Polymer

<tbody> </tbody>
Reagents (f. 20 µL)
Purified Monomer A 10 µL
Purified Monomer B 10 µL

Procedure

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

2) Each Monomer solution was purified by spin column.

3) The reagents was 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="2-1-3"> </a>



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

<a name="2-2"> </a>



2-2. The Motor in the liposome

<a name="2-2-1"> </a>



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

<tbody> </tbody>
Reagents (f.1420 µL)
Lipid mix (f. 0.5 mM)¹ 400 µL
Glucose (f. 200 mM) 500 µL
Emulsion mix² 520 µL
<tbody> </tbody>
¹Lipid mix (f. 3 mM)
POPC 12 mg
Paraffin 5 mL
<tbody> </tbody>
²Emulsion mix
Lipid mix (f. 0.5mM) 500 µL
Inner solution³ 10 µL
The Monomer 10 µL
<tbody> </tbody>
³Inner solution
HEPES (f. 50 mM) 250 µL
Sucrose(f. 400 mM) 250 µL
Pyranine 2 µL

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.



<a name="2-2-2"> </a>



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

<a name="2-2-3"> </a>



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




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