Name: in vitro assembly of semi-artificial molecular machine and its use for detection of dna damage
Uploaded: 2012-01-11T13:00:00
Description: Watch this Scientific Journal Video about In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage at JoVE.com

这项研究得到了授予R01NS062842国家神经紊乱和中风研究所，国立卫生研究院（VVD）和国立神经疾病和中风，国家卫生研究院通过复苏法案（VVD）和R21的R21 NS064403补助EB006301国家生物医学成像和生物工程研究所，国立卫生（VVD）研究院。

分子机器的检测部分，首先是因为他们的准备需要更多的时间比分子器件的装配，应准备。兴建工程，以及5 -6μm厚的部分切多聚甲醛固定，石蜡包埋组织块。使用幻灯片的品牌如ProbeOn另外收取和precleaned幻灯片“（Fisher Scientific这）或类似的保留部分，。我们建议在第一次使用与知名的DNA损伤的模式，其中包含两个DNA酶I -和DNase II型断裂，如地塞米松治疗的细胞凋亡大鼠胸腺13,14 ，一个组?...

<ol>
	<li>Meacham, G. C., Patterson, C., Zhang, W., Younger, J. M., Cyr, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nature. Cell. Biol. 3, 100-105 (2001).</li><li>Schuldt, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+Pol+expeditions.">Dynamic Pol expeditions.</a> Nature. Cell. Biol. 4, E279-E279 (2002).</li><li>Urry, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+machines:+how+motion+and+other+functions+of+living+organisms+can+result+from+reversible+chemical+changes.">Molecular machines: how motion and other functions of living organisms can result from reversible chemical changes.</a> Angew. Chem. Int. Ed. Engl. 32, 819-841 (1993).</li><li>Schneider, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theory+of+molecular+machines+I.+Channel+capacity+of+molecular+machines.">Theory of molecular machines I. Channel capacity of molecular machines.</a> J. Theor. Biol. 148, 83-123 (1991).</li><li>McClare, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+machines,+Maxwell's+demon+and+living+organisms.">Chemical machines, Maxwell's demon and living organisms.</a> J. Theor. Biol. 30, 1-34 (1971).</li><li>Spirin, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribosome+as+a+molecular+machine.">Ribosome as a molecular machine.</a> FEBS Lett. 514, 2-10 (2002).</li><li>Fabbrizzi, L., Foti, F., Licchelli, M., Maccarini, P. M., Sacchi, D., Zema, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-emitting+molecular+machines:+pH-induced+intramolecular+motions+in+a+fluorescent+nickel(II)+scorpionate+complex.">Light-emitting molecular machines: pH-induced intramolecular motions in a fluorescent nickel(II) scorpionate complex.</a> Chemistry. 8, 4965-4972 (2002).</li><li>Drexler, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nanosystems: Molecular Machinery, Manufacturing and Computation. , (1992).</li><li>Freitas, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nanomedicine: basic capabilities. , (1999).</li><li>Didenko, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+probes+using+fluorescence+resonance+energy+transfer+(FRET):+designs+and+applications.">DNA probes using fluorescence resonance energy transfer (FRET): designs and applications.</a> BioTechniques. 31, 1106-1121 (2001).</li><li>Minchew, C. L., Didenko, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+probes+detecting+the+phagocytic+phase+of+apoptosis:+enzyme-substrate+complexes+of+topoisomerase+and+DNA.">Fluorescent probes detecting the phagocytic phase of apoptosis: enzyme-substrate complexes of topoisomerase and DNA.</a> Molecules. 16, 4599-4614 (2011).</li><li>Didenko, V. V., Minchew, C. L., Shuman, S., Baskin, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Semi-artificial+fluorescent+molecular+machine+for+DNA+damage+detection.">Semi-artificial fluorescent molecular machine for DNA damage detection.</a> Nano. Letters. 4, 2461-2466 (2004).</li><li>Didenko, V. V., Hornsby, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presence+of+double-strand+breaks+with+single-base+3'+overhangs+in+cells+undergoing+apoptosis+but+not+necrosis.">Presence of double-strand breaks with single-base 3' overhangs in cells undergoing apoptosis but not necrosis.</a> J. Cell Biol. 135, 1369-1376 (1996).</li><li>Didenko, V. V., Tunstead, J. R., Hornsby, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biotin-labeled+hairpin+oligonucleotides.+Probes+to+detect+double-strand+breaks+in+DNA+in+apoptotic+cells.">Biotin-labeled hairpin oligonucleotides. Probes to detect double-strand breaks in DNA in apoptotic cells.</a> Am. J. Pathol. 152, 897-902 (1998).</li><li>Jones, R. A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Soft Machines: Nanotechnology and life. , (2007).</li><li>Samejima, K., Earnshaw, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trashing+the+genome:+Role+of+nucleases+during+apoptosis.">Trashing the genome: Role of nucleases during apoptosis.</a> Nat. Rev. Mol. Cell. Biol. 6, 677-688 (2005).</li></ol>

在这段视频中，我们将演示如何组装和使用双标签的DNA损伤传感器。传感器是一个由生物分子发动机，病毒编码的蛋白质VACC TOPO的人工组件联系在一起驱动的分子机器。所提出的发展充分体现了一个生物功能的做法，主张适应的无毒分子尺度器件12,15设计的生物结构，架构和实际零件和电池组件。这种方法可以解决两个问题的内在体内的纳米传感器的领域： 1。使用传统的纳米材料?...

<ol><li>二甲苯</li><li> 80％和96％的乙醇。 </li><li> 2％的蒸馏水中牛血清白蛋白（BSA）的解决方案。 </li><li>寡核苷酸1：双簪，双荧光素和四甲标记： </li></ol> 5&#39; - AAG GGA CCT的气相色谱F GCA的GGT的CCC认证TTA ACG的CAT R在GCG TT - 3&#39;，F - FITC - DT，R -四甲- DT。其他红移荧光团，如肾上腺素TR，罗丹明或TAMRA，可以用来代替四甲为R。 另外，您可以使用寡核苷酸2 - 双发夹单（单一类型的DNA断裂（DNA酶II型只）检测用荧光素标记的单荧光携带探针相当便宜，方便，每当单一DNA断裂的类型必须标明：5&#39; - AAG GGA CCT的GC F GCA的GGT的CCC认证TTA ACG的CAT ATG总工会的T - 3&#39;，F - FITC - DT </p&gt; <ol start="5"><li>寡核苷酸3。钝端罗丹明标记的发夹，在T4 DNA连接酶的原位结扎信号增强：5&#39; - GCG GAC中注册税务师彗星 R G GTC豪CGC - 3&#39;，R -四甲- DT </li><li>痘苗病毒DNA拓扑异构酶（TOPO VACC）升 - 3000 U /μL（生动的技术）。在最初的实验中，我们使用215％每25μL的反应组合VACC TOPO pmoles（7.1微克）。然而，拓扑异构酶浓度可显着降低，而不损失灵敏度。后来我们用了类似的结果四次少量的VACC TOPO（1.76微克）在25μL的反应混合物中每节每节。减少880纳克（25μL的反应组合）的酶量在信号较弱VACC TOPO和8.8纳克，导致生产没有检测到信号。 </li><li> T4 DNA连接酶5 U /μL（罗氏公司）。这种高度集中的连接酶制剂提供了在本实验条件下的最佳信号。 </l我&gt; <li> 10 × T4 DNA连接酶反应缓冲液：660毫米，50毫米的Tris - HCl MgCl 2的 10毫米（ATP，pH值7.5，10毫米dithioerythritol，20℃）（罗氏公司）。 ATP的反应缓冲液很容易被破坏，在解冻冷冻的重复周期。分装在15-20微升小部分，并存储在缓冲区 - 20 ° C使用一次。 </li><li> bidistilled水PEG - 8000（Sigma公司）的30％（W / V）的解决方案。 15％PEG - 8000在反应混合物强烈刺激的检测，其成员的有效浓度增加量排斥。 </li><li>蛋白酶K（罗氏）20 mg / ml的蒸馏水原液。储存在 - 20 ° C反应使用50mg/mL的解决方案，在PBS准备从股市。不要重复使用。 </li><li>用DAPI（媒介实验室）Vectashield。 </li><li>碳酸氢钠缓冲液：50米中号碳酸氢钠3，15M M氯化钠，pH值8.2。 </li><li> 22x22mm或22x40mm的玻璃或塑料盖玻片。塑料盖玻片是可取的，在反应过程中，他们一个重新容易从一节中删除。 </li></ol>

in vitro assembly of semi-artificial molecular machine and its use for detection of dna damage

自然发生的生物分子机器工作在每一个活细胞，并显示各种设计1-6。然而，人工分子机器设备上，即分子马达定向运动的能力，以及按比例缩小的机械部件（车轮，axels，吊坠等）7-9的中心发展。这模仿的宏观机，这些设备的基本的物理性质，化学性质，如惯性和动量守恒，即使不是在10纳米世界环境使用。替代设计，不按机械macromachines计划和使用生物分子的进化发展的机制，可以利用纳米世界的具体条件优势。此外，适应实际的生物分子纳米设计的目的，降低了纳米技术的产品可能会造成的潜在危险。在这里，我们展示了这样一个启用生物构造的组装和应用，EMI人工分子器件相结合的人工组件的一个自然发生​​的分子机器。从酶学点来看，我们的结构是一个设计师荧光酶 - 底物复合物放在一起执行特定的有用的功能。这个大会是由定义的分子机器，因为它包含一个 12 。然而，其一体化工程的一部分-荧光双发夹-重新定向到一个新的任务标签DNA损伤12。 我们的构造，组装出32 MER DNA和一种酶的牛痘拓扑异构酶I（VACC TOPO）。本机，然后使用自己的材料来制造两个荧光标记的检测器（图1）。单位之一（绿色荧光）进行VACC TOPO的共价连接到其3&#39;end和另一个单位（红色荧光），是同一个终端3&#39;OH发夹。的单位是短命的，并迅速重新组合成原来的构造，随后recleaves。在DNA的情况下，打破了这两个循环的方式不断独立和religate单位。在与DNA损伤，探测器单元的拓扑异构酶选择性重视5&#39;OH（DNA酶II型断裂）11,12平末端的DNA断裂，荧光标签的组织切片。第二，酶寡核苷酸裂解后形成的发夹，将结扎一个5&#39;PO 4钝端突破（DNA酶I型断裂）11,12，T4 DNA连接酶是在解决方案 13,14中。当T4 DNA连接酶将被添加到一个组织部分或溶液中的DNA与5&#39;PO 4休息钝端，连接酶与5&#39;PO 4 DNA末端反应，形成半稳定酶- DNA复合物。钝端发夹将与这些复合物释放的DNA连接酶和共价连接的发夹，从而标签5&#39;PO 4平末端的DNA断裂。 这方面的发展充分体现了新的切实可行的办法，以日E的分子机器的设计，并提供有用的一个固定的细胞和组织的细胞凋亡和DNA损伤检测传感器。

Watch this Scientific Journal Video about In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage at JoVE.com

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

我们证明由拓扑异构酶蛋白驱动的分子尺度器件的组装和应用。构造是一个标签由组织切片中的DNA断裂的两个主要类型，其两端连接两个不同的荧光团的生物分子传感器。

在检测DNA损伤的体外半人工分子机器，其使用的大会

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs 1-6. Yet the development of artificial molecular ...

Research

JoVE Journal

Bioengineering

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

无试剂检测核酸，蛋白质和小分子的电化学DNA生物传感器的制备

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

科研

生物工程

Synthesis, Assembly, and Characterization of Monolayer Protected Gold Nanoparticle Films for Protein Monolayer Electrochemistry

单层的合成，装配和表征蛋白质单分子膜的电化学保护的金纳米粒子薄膜

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently ...

CometChip: A High-throughput 96-Well Platform for Measuring DNA Damage in Microarrayed Human Cells

CometChip：一种高通量的96孔平台的微阵列人类细胞测量DNA损伤

DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular ...

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

使用高通量的<em&gt;在体外</em&gt;微流控系统开发口服多物种生物膜

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

眼刺激试验（EIT）使用重构型人角膜上皮样（RHCE）组织模型眼刺激性化学品危险性鉴定

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Micropatterning and Assembly of 3D Microvessels

缩微和3D微血管大会

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

模块化 DNA 设备的自动机器人液体处理组件

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

Synthesis of Cationized Magnetoferritin for Ultra-fast Magnetization of Cells

对细胞的超快磁化阳离子化Magnetoferritin的合成

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide ...

Construction and Use of an Electrical Stimulation Chamber for Enhancing Osteogenic Differentiation in Mesenchymal Stem/Stromal Cells In Vitro

电刺激室的构建与应用, 促进间充质茎间质细胞体外成骨分化

Mesenchymal stem/stromal cells (MSCs) have been used extensively to promote bone healing in tissue engineering approaches. Electrical stimulation (EStim) ...

Bioparticle Microarrays for Chemotactic and Molecular Analysis of Human Neutrophil Swarming in vitro

人体神经细胞在体外热的化学和分子分析的生物粒子微阵列

Neutrophil swarming is a cooperative process by which neutrophils seal off a site of infection and promote tissue reorganization. Swarming has classically ...