Name: spatiotemporally controlled nuclear translocation of guests in living cells using caged molecular glues as photoactivatable tags
Uploaded: 2019-01-17T13:00:00
Description: Watch this Scientific Journal Video about Spatiotemporally Controlled Nuclear Translocation of Guests in Living Cells Using Caged Molecular Glues as Photoactivatable Tags at JoVE.com

我们感谢东京大学纳米生物集成中心。这项工作得到了青年科学家补助金 (b) (26810046) 至 k. o. 的支持, 并得到了对 t. a. r. m. 的特别促进研究补助金 (25000005) 的部分支持, 感谢日本促进科学协会研究研究研究研究研究研究研究研究研究研究研究金 (jps)。), 为青年科学家和主要研究生院 (gplli) 计划。

1. 使用笼子胶粘剂 r 标签的客人的准备
<ol><li>准备笼子里的 glu-nbd 解决方案。<ol>
<li>按照前面描述的过程合成笼子 gnbd (图 1) 20。</li>
		<li>制备干二甲基亚硫酸盐 (dmso) 中的环胶-nbd (10 mm) 的库存溶液。 注意: 将库存解决方案存储在黑暗中。该溶液可在使用时用水缓冲液或细胞培养介质稀释。</li>
	</ol>
</li>
	<li>准备笼</s...

<ol>
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K., Okuro, K., Sasaki, S., Hoashi, Y., Aida, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reductively+Cleavable+Nanocaplets+for+siRNA+Delivery+by+Template-Assisted+Oxidative+Polymerization.">Reductively Cleavable Nanocaplets for siRNA Delivery by Template-Assisted Oxidative Polymerization.</a> Journal of the American Chemical Society. 137, 15608-15611 (2015).</li><li>Hatano, J., Okuro, K., Aida, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoinduced+Bioorthogonal+1,3-Dipolar+Poly-cycloaddition+Promoted+by+Oxyanionic+Substrates+for+Spatiotemporal+Operation+of+Molecular+Glues.">Photoinduced Bioorthogonal 1,3-Dipolar Poly-cycloaddition Promoted by Oxyanionic Substrates for Spatiotemporal Operation of Molecular Glues.</a> Angewandte Chemie, International Edition. 55, 193-198 (2016).</li><li>Arisaka, A., Mogaki, R., Okuro, K., Aida, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caged+Molecular+Glues+as+Photoactivatable+Tags+for+Nuclear+Translocation+of+Guests+in+Living+Cells.">Caged Molecular Glues as Photoactivatable Tags for Nuclear Translocation of Guests in Living Cells.</a> Journal of the American Chemical Society. 140, 2687-2692 (2018).</li><li>Suzuki, Y., Okuro, K., Takeuchi, T., Aida, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Friction-Mediated+Dynamic+Disordering+of+Phospholipid+Membrane+by+Mechanical+Motions+of+Photoresponsive+Molecular+Glue:+Activation+of+Ion+Permeation.">Friction-Mediated Dynamic Disordering of Phospholipid Membrane by Mechanical Motions of Photoresponsive Molecular Glue: Activation of Ion Permeation.</a> Journal of the American Chemical Society. 134, 15273-15276 (2012).</li><li>Wang, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-water-content+mouldable+hydrogels+by+mixing+clay+and+a+dendritic+molecular+binder.">High-water-content mouldable hydrogels by mixing clay and a dendritic molecular binder.</a> Nature. 463, 339-343 (2010).</li><li>Tamesue, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linear+versus+Dendritic+Molecular+Binders+for+Hydrogel+Network+Formation+with+Clay+Nanosheets:+Studies+with+ABA+Triblock+Copolyethers+Carrying+Guanidinium+Ion+Pendants.">Linear versus Dendritic Molecular Binders for Hydrogel Network Formation with Clay Nanosheets: Studies with ABA Triblock Copolyethers Carrying Guanidinium Ion Pendants.</a> Journal of the American Chemical Society. 135, 15650-15655 (2013).</li><li>Mohr, D., Frey, S., Fischer, T., G&#252;ttler, T., G&#246;rlich, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterisation+of+the+passive+permeability+barrier+of+nuclear+pore+complexes.">Characterisation of the passive permeability barrier of nuclear pore complexes.</a> EMBO Journal. 28, 2541-2553 (2009).</li><li>Best, M. 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以前使用 cagednls 7、8、9实现了对光触发蛋白质转移到细胞核中的研究。如前所述, 这些方法需要额外的技术, 以纳入 nls 标记的蛋白质到细胞质。相反, 我们的 c蒂胶粘剂-r 标签不仅可以实现光致核的移位, 还可以让客人的手机吸收。笼胶- r标记的这一特性有利于在体内应用。
笼子...

携带遗传信息的细胞核是作为亚细胞药物传递靶点最重要的细胞器之一, 因为基因复制和表达的调节对治疗包括癌症和基因在内的各种疾病是有效的疾病1,2,3。对于核输送药物, 核定位信号 (nls)4、5、6 等肽标记的共轭已被广泛研究。然而, 为了减少不想要的副作用, 对核转移进行时空控制是必要的。
以前, 利用笼子 nls7、8、9实现了蛋白质在细胞核中的光触发移位。nls 通过与细胞质转运蛋白结合而迁移到细胞核中.在所报道的方法中, 采用遗传密码扩展技术 9, 通过微注射8将带有冠状 nls 的客人蛋白直接整合到细胞质中, 或在目标细胞中表达。因此, 一种既能实现细胞吸收又能实现光致核转移的方法, 有利于实际应用。
在此, 我们描述了光触发核移位的客人在活细胞使用树突状胶-r,图 1)标签活细胞。水溶性分子胶10,11,12,13, 14, 15,16, 17,18,19,20,21,22,23轴承多个 gu+吊坠以前已经开发, 其中紧密粘附蛋白质11,12,13,14,15, 16,17,核酸18,19,20, 磷脂膜21, 和粘土纳米板22,23通过在目标上的 gu+挂件和氧阴离子基团之间形成多个盐桥。笼胶 r的 gu +吊坠受阴离子光裂解组丁酸取代的硝基氮氧基 (banvoc) 的保护。贴有笼状胶 r 标记的客人通过内吞进入活细胞, 并留在内染色体中 (图 2)。在光照射下, c形胶-r 的banvoc 基团分离出一种不带多个 gu+吊坠的未分类分子胶 (解链胶 r), 从而促进了标记客人的迁移进入细胞质, 然后是细胞核 (图 2)。通过暴露在 uv 或双光子近红外 (nir) 光下, 可以在不产生严重光毒性的情况下, 取消笼子胶-r 标记。我们用量子点 (qd) 和荧光染料 (硝基苯并二恶唑), 用胶格 r 标记演示了高分子嘉宾和小分子嘉宾的时空控制核输送;(nbd), 分别作为例子。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58631/58631fig1.jpg"> 图 1:笼胶-r 的原理图结构.环胶 r 的9个鸟离子 (gu+) 吊坠受丁酸取代的硝基硝基氧氧氧烷基 (banvoc) 保护。banvoc 基团通过紫外或双光子近红外光的照射进行切割。用硝基苯二唑 (nbd) 或二苯环磷酰胺 (dbco) 对环胶-r 的焦核进行了功能化处理。在引用20的允许下重新打印。<a href="https://www.jove.com/files/ftp_upload/58631/58631fig1large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58631/58631fig2.jpg"> 图 2: 与带笼子胶粘剂 r 标记共轭的来宾的光触发核移位示意图.通过内吞将客魔/笼胶-r 共轭带入活细胞。在光照射下,笼子胶质 r 标签被取消, 以产生一个取消胶-r 标签, 这可以促进标记的客人的内部染色体逃逸。随后, 贴有标记的客人迁移到细胞核中。在引用20的允许下重新打印。<a href="https://www.jove.com/files/ftp_upload/58631/58631fig2large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>

<table>
	<tbody>
		<tr>
			<td>Azide-PEG4-NHS ester</td>
			<td>Click Chemistry Tools</td>
			<td>AZ103</td>
			<td></td>
		</tr>
		<tr>
			<td>Q-dot 655 ITK</td>
			<td>Invitrogen</td>
			<td>Q21521MP</td>
			<td></td>
		</tr>
		<tr>
			<td>Regenerated cellulose membrane (MWCO 3,500)</td>
			<td>NIPPON Genetics</td>
			<td>TOR-3K</td>
			<td></td>
		</tr>
		<tr>
			<td>Regenerated cellulose membrane (MWCO 25,000)</td>
			<td>Harvard Apparatus</td>
			<td>7425-RC25K</td>
			<td></td>
		</tr>
		<tr>
			<td>Hep3B Cells</td>
			<td>ATCC</td>
			<td>HB-8064</td>
			<td></td>
		</tr>
		<tr>
			<td>8-chambered glass substrate</td>
			<td>Nunc</td>
			<td>155411JP</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well culture plate</td>
			<td>Nunc</td>
			<td>167008</td>
			<td></td>
		</tr>
		<tr>
			<td>Eagle&#39;s minimal essential medium (EMEM)</td>
			<td>Thermo Fisher Scientific</td>
			<td>10370-021</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>GE Healthcare</td>
			<td>SH30406.02</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate buffer saline (D-PBS)</td>
			<td>Wako Pure Chemical Industries</td>
			<td>045-29795</td>
			<td></td>
		</tr>
		<tr>
			<td>LysoTracker Red</td>
			<td>Lonza Walkersville</td>
			<td>PA-3015</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Dojindo</td>
			<td>H342</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Counting Kit-8</td>
			<td>Dojindo</td>
			<td>CK04</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal laser scanning microscope</td>
			<td>Carl-Zeiss</td>
			<td>LSM 510</td>
			<td>Equipped with two-photon excitation laser (Mai Tai laser, Spectra-Physics)</td>
		</tr>
		<tr>
			<td>Confocal laser scanning microscope</td>
			<td>Leica</td>
			<td>TCS SP8</td>
			<td></td>
		</tr>
		<tr>
			<td>Xenon light source</td>
			<td>Asahi Spectra</td>
			<td>LAX-102</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>Molecular Devices</td>
			<td>SpectraMax Paradigm</td>
			<td></td>
		</tr>
	</tbody>
</table>

spatiotemporally controlled nuclear translocation of guests in living cells using caged molecular glues as photoactivatable tags

细胞核是作为亚细胞药物传递靶点的最重要的细胞器之一, 因为基因复制和表达的调节对治疗各种疾病是有效的。在这里, 我们展示了光触发核移位的客人使用笼分子胶 (笼胶 r) 标签, 其多个鸟苷离子 (gu+) 挂件是由一个阴离子光裂解组 (丁酸酯取代硝基甲氧基酯;文学学士nvoc)。贴有笼状胶 r 标记的客人通过内吞进入活细胞, 并留在内质体中。然而, 在光辐射下,笼胶-r 被转化为不带多个 gu+吊坠的未分类分子胶 (Uncaged-r), 便于客人的内部逃逸和随后的核移位。这种方法对于现场选择性核靶向药物输送很有希望, 因为标记的客人只有在光辐照时才能迁移到细胞质中, 然后是细胞核。笼中胶粘剂 r 标签可以提供大分子客人, 如量子点 (qd) 以及小分子客人。笼中胶-r 标签不仅可以用紫外光照射, 还可以用双光子近红外 (nir) 光进行解锁, 从而深入组织。

在光照射前, 用笼状葡萄-nbd 培养的 hep3b 细胞从其内部显示出点状荧光发射 (ext = 488 nm;图 4 a 和4A, 绿色)。在543纳米的激发下, 红荧光染料 (图 4b和4B, 红色) 得到了类似的显微图像, 表明笼子葡萄-nbd 在内质体中局部化。因此, 在细胞核外观察到可分配给cagedglar-nbd (<strong clas...

Watch this Scientific Journal Video about Spatiotemporally Controlled Nuclear Translocation of Guests in Living Cells Using Caged Molecular Glues as Photoactivatable Tags at JoVE.com

Spatiotemporally Controlled Nuclear Translocation of Guests in Living Cells Using Caged Molecular Glues as Photoactivatable Tags

该协议描述了使用笼子分子胶标签的光触发核移位的客人在活细胞中的位置。该方法对现场选择性核靶向药物的输送具有广阔的应用前景。

利用笼子分子胶作为可活性标记的活细胞中的贵宾的时空控制核移位

The cell nucleus is one of the most important organelles as a subcellular drug-delivery target, since modulation of gene replication and expression is ...

此方法可以帮助回答药物输送领域的关键问题，例如如何向特定的细胞粒细胞提供药物。该技术的主要优点是气体的核转移可以通过光引起。演示程序将是丽娜 · 莫加基和秋叶子 · 阿里萨卡， 我小组的毕业生。开始准备量子点链接笼分子胶通过合成笼状分子胶链接二苯环环素，如在其他地方更详细地描述。在干DMSO中准备笼状分子胶连接二苯并环环的10毫摩尔库存溶液。然后准备量子点链接笼状分子胶，首先使阿西眼功能化量子点。为此，在 DMF 中向包含 500 纳米摩尔的量子点的 DMF 溶液中添加 100 微升 125 微摩尔 aseye PEG4 NHS 酯，这些溶液涂有胺功能化挂钩。在室温下搅拌混合物一小时。将所得溶液放在再生的纤维素膜中，对800毫升DMF进行24小时透析。接下来，将笼状分子胶的库存溶液与DDMF连接50微摩尔，并在透析后溶液中加入200微升。在室温下搅拌混合物三小时。然后使用再生的纤维素膜对800毫升新鲜DMF进行24小时对，分子量切断25000。24 小时后，用 DMF 将所得溶液稀释至 200 纳米摩尔。在标准培养条件下，在 Eagle 的最小基本介质中维护人类肝细胞癌 HEP3 B 细胞，其中含有 10%FBS。实验前一天，将5000个 HEP3 B 细胞的种子在 200 微升培养基中放入 8 室玻璃滑梯的每个井中。然后盖住幻灯片，将其放入孵化器 24 小时。第二天取出培养培养剂，用100微升预热PBS冲洗细胞两次。在8室滑轨的每个井中加入200微升的FBS自由介质，其中含有10微摩尔的荧光染色笼状分子胶。孵育3小时后，取出培养培养剂，用100微升PBS冲洗细胞样品两次。为了可视化，内糖体添加200微升培养培养媒体，其中包含100纳米摩尔的额外红色荧光染料，如LysoTracker Red。孵育产生的细胞样本20分钟。然后取出培养介质，用100微升PBS冲洗细胞样品两次。之后，将细胞返回培养培养的200微升。为了诱导荧光染色笼状分子胶的核转移，将细胞放在装有365纳米带通滤波器的100瓦Xenon光源下。将细胞暴露在紫外线下 2 分钟。在此期间，保持控制细胞样本在黑暗中无紫外线照射。玻璃基板的盖子可以取下，以有效进行紫外线照射，但要小心，因为长时间暴露在紫外线下可能会导致细胞死亡。为了可视化，核在培养基中加入一个微升的霍斯特染色剂。孵育产生的细胞样本10分钟。然后将样品放在共和激光扫描显微镜的舞台上。记录荧光染色笼状分子胶、LysoTracker Red 和核污渍的显微图。如前所述，种子5000人类肝细胞癌 HEP3 B 细胞进入每个井的 8 井室滑动和培养在标准条件下 24 小时。向细胞样品提供含有荧光染色笼状分子胶的200微升FBS自由培养介质。孵育产生的细胞样本3小时。然后取出培养介质，用100微升PBS冲洗细胞样品两次。将细胞返回200微升的培养介质。然后接受样品共和激光扫描显微镜，并记录下所附文本协议中描述的显微图。如前所述，种子5000人肝细胞癌 HEP3 B 细胞进入每个井的 8 井室滑动和培养在标准条件下 24 小时。向细胞样品提供200微升FBS自由培养介质，其中含有10纳米摩尔的量子点链接笼状分子胶。孵育产生的细胞样本3小时。然后取出介质，用 100 微升 PBS 冲洗细胞样品两次。冲洗细胞后，将它们返回到培养基的200微升。然后将板放在配备 365 纳米带通滤波器的 100 瓦 Xenon 光源下。将细胞样品暴露在紫外光下 2 分钟，使控制样品保持黑暗。为了可视化，核在培养基中加入1微升的霍赫斯特染色剂。孵育产生的细胞样本10分钟。然后使用共和激光扫描显微镜对细胞进行成像。在实验前一天，将人类肝细胞癌 HEP3 B 细胞以每井 5000 个细胞种子到 96 井培养板中。然后用200微升培养的培养剂覆盖细胞，然后将盘子放在培养箱中24小时。第二天取出培养培养剂，用100微升PBS冲洗细胞样品两次。然后加入200微升FBS自由培养介质，含有荧光染色笼状分子胶，浓度在0.1至100微摩尔。孵育板3小时，然后将板放在UV光源下。将细胞样品暴露在紫外光下 2 分钟，使控制样品保持黑暗。在细胞计数试剂的10微升下紫外线照射到培养基，并孵育产生的细胞样本2小时。然后使用微孔板读卡器读取样品在 450 纳米的吸收量。在照片辐射之前，用荧光染色笼状分子胶孵育的 HEP3 B 细胞从内部显示双核荧光发射，一个类似的显微图显示 LysoTracker Red 表明荧光染色笼状分子胶在内含体中局部化。因此，在紫外线辐射之前，在细胞核外观察到可分配给荧光染色笼状分子胶的荧光发射。紫外线辐射后，荧光表明荧光染色笼状分子胶被解开并迁移到细胞质和细胞核。这种荧光染色笼状分子胶的核翻译可以通过近红外光选择性地诱导。在非辐照区域，荧光染色的笼状分子胶没有从内分体中逸出，并且仍然作为双核荧光。树突笼状分子胶标签也可以将大分子像量子点等大分子进入细胞核。这些结合物可以进入细胞，在紫外线照射2分钟后，量子点的荧光发射可以在细胞核内看到。这一技术得到发展，为基因治疗领域的研究人员探索低副作用治疗铺平了道路。

Research

JoVE Journal

Chemistry

Facile and Efficient Preparation of Tri-component Fluorescent Glycopolymers via RAFT-controlled Polymerization

通过RAFT控制聚合轻便和高效制备三组分荧光Glycopolymers

Synthetic glycopolymers are instrumental and versatile tools used in various biochemical and biomedical research fields. An example of a facile and ...

科研

化学

Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation

放射性标记和磷酸肌醇的细胞水平的定量高效液相色谱耦合流动闪烁

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular ...

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical (Methylation) and Physical (Mass Spectrometry, Nuclear Magnetic Resonance) Techniques

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical Methylation and Physical Mass Spectrometry, Nuclear Magnetic Resonance Techniques

使用酶法，化学（甲基化）和物理（质谱，核磁共振）技术，植物墙Heteroxylans测序

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of ...

Investigations on the Ga(III) Complex of EOB-DTPA and Its 68Ga Radiolabeled Analogue

Investigations on the GaIII Complex of EOB-DTPA and Its 68Ga Radiolabeled Analogue

在EOB-DTPA和镓（三）复杂的调查其<sup&gt; 68</sup&gt;嘎放射性标记模拟

We demonstrate a method for the isolation of EOB-DTPA (3,6,9-triaza-3,6,9-tris(carboxymethyl)-4-(ethoxybenzyl)-undecanedioic acid) from its Gd(III) ...

Characterizing Electron Transport through Living Biofilms

通过活生物膜表征电子传输

Here we demonstrate the method of electrochemical gating used to characterize electrical conductivity of electrode-grown microbial biofilms under ...

Laser-heating and Radiance Spectrometry for the Study of Nuclear Materials in Conditions Simulating a Nuclear Power Plant Accident

核材料在核电厂事故模拟条件下的激光加热和辐射光谱研究

Major and severe accidents have occurred three times in nuclear power plants (NPPs), at Three Mile Island (USA, 1979), Chernobyl (former USSR, 1986) and ...

In Situ Detection and Single Cell Quantification of Metal Oxide Nanoparticles Using Nuclear Microprobe Analysis

In Situ Detection and Single Cell Quantification of Metal Oxide Nanoparticles Using Nuclear Microprobe Analysis

原位金属氧化物纳米粒子的核探针检测与单细胞定量研究

Micro-analytical techniques based on chemical element imaging enable the localization and quantification of chemical composition at the cellular level. ...

Controlled Photoredox Ring-Opening Polymerization of O-Carboxyanhydrides Mediated by Ni/Zn Complexes

Controlled Photoredox Ring-Opening Polymerization of O-Carboxyanhydrides Mediated by Ni/Zn Complexes

镍/锌配合物介导的O-Carboxyanhydrides 的控制 Photoredox 环开聚合

Here, we describe an effective protocol that combines photoredox Ni/Ir catalysis with the use of a Zn-alkoxide for efficient ring-opening polymerization, ...

Achieving Moderate Pressures in Sealed Vessels Using Dry Ice As a Solid CO2 Source

Achieving Moderate Pressures in Sealed Vessels Using Dry Ice As a Solid CO2 Source

用干冰作为固体 CO2源实现密封容器中的适度压力

Herein is presented a general strategy to perform reactions under mild to moderate CO2 pressures with dry ice. This technique obviates the need for ...

Light-driven Molecular Motors on Surfaces for Single Molecular Imaging

表面光驱动分子电机的单分子成像

The design and synthesis of a synthetic system that aims for the direct visualization of a synthetic rotary motor at the single molecule level on surfaces ...