Editing XIE/EnZyme:2025/Oct

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== Protein(RuCHMO/GDH) Conjugation to DNA Origami ==
=== Methods ===
=== Conjugation ===
*''' Proteins containing 6x Histidine at the C terminus reacted, with the chemical DBCO-PEG4-Bis-sulfone(DPBS) for 4 h at room temperature. The bis-sulfone group reacts with the Histidines at the C terminus of the protein, and then an azide modified oligonucleotide is conjugated to the protein by click chemistry between the azide- and the DBCO-group.'''

=== Incubation ===
*'''conjugation: DNA Origami = 20:1, and incubate in a PCR machine with a temperature ramp starting from 1 h at 37 ℃ follow by 14 h at 22 ℃, and immediately after incubation the nanopatterns are stored at 4 ℃.'''
=== Oligonucleotide Sequence ===
*'''sequence(RuCHMO):CTCTCCTTCTTCCCTTTCTTT'''
*'''Sequence(GDH):TTCGACAGCATGAACATCAGC'''
=== Protein ===
*'''RuCHMO: 65.45 KDa'''
*'''GDH: 35 KDa'''

=== Purification ===
*''' 30 kDa MWCO 0.5 ml Amicon centrifugal filters (Milli-pore)'''
=== electro-phoresis ===
{| class="wikitable"
|+ Sample
|-
! Reagent
! Sequence
|-
| DBCO-PEG4-Bis-sulfone
| 
|-
| azide modified oligonucleotide
| CTCTCCTTCTTCCCTTTCTTT
|-
| azide modified oligonucleotide
| TTCGACAGCATGAACATCAGC
|}
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== Protocol (版本 1.0) ==
=== 1. GDH 制备 ===
取 '''17.5 μg GDH''' 溶解于 '''25 μL 1×PBS''' 中。

=== 2. 稀释 GDH（样品液） ===
{| class="wikitable" style="text-align:center; width:80%"
! 样品 !! 原样品浓度 !! 终样品浓度 !! 加入量 !! 总体积
|-
| GDH || 0.7 mg/mL || 0.175 mg/mL || 25 μL || 100 μL
|}

=== 3. 溶解 DBCO-PEG4-Bis-Sulfone 于 DMF(N,N-二甲基甲酰胺) 中（100 mM） ===

=== 4. 稀释 DBCO（样品液） ===
{| class="wikitable" style="text-align:center; width:80%"
! 样品 !! 原样品浓度 !! 终样品浓度 !! 加入量 !! 总体积
|-
| DBCO || 100 mM || 20 mM || 2 μL || 10 μL
|}


=== 5. 向 GDH 样品液中加入 DBCO，并在室温下孵育 4 h ===
{| class="wikitable" style="text-align:center; width:90%"
! 样品 !! 最终浓度 !! 分子量 !! 总摩尔量 !! Eq DBCO !! DBCO 摩尔量 !! DBCO 加入量 !! 总体积
|-
| GDH || 5 mM || 35 kDa || 0.5 nMol || 20 Eq || 10 nMol || 0.5 μL || 100 μL
|}

=== 6. 使用 10 kDa 超滤管超滤去除游离 DBCO ===


=== 7. 加入 Oligo，并在 37 ℃ 下孵育 4 h ===
{| class="wikitable" style="text-align:center; width:90%"
! 样品 !! 总摩尔量 !! Eq Oligo !! Oligo 摩尔量 !! Oligo 加入量 !! 总体积
|-
| GDH || 0.5 nMol || 20 Eq || 10 nMol || 0.5 μL || 100 μL
|}

=== 8. 凝胶电泳 ===
等量的 '''GDH''' , '''偶联产物''' 和 '''Marker''' 跑胶，对蛋白进行'''考马斯亮蓝'''染色比对。