在检测DNA损伤的体外半人工分子机器，其使用的大会
Summary
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。
Abstract
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'end和另一个单位（红色荧光），是同一个终端3'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'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'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'PO 4休息钝端，连接酶与5'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'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。
Protocol
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
- Xylene
- 80 and 96% Ethanol.
- 2% solution of bovine serum albumin (BSA) in distilled water.
- Oligonucleotide 1: a double-hairpin, dual labeled with fluorescein and tetramethylrhodamine:
5′-AAG GGA CCT GCF GCA GGT CCC TTA ACG CAT RAT GCG TT- 3′; F – FITC-dT; R – Tetramethylrhodamine-dT. Other red-shifted fluorophores such as BODIPY TR, rhodamine or TAMRA can be used instead of tetramethylrhodamine as R.
Alternatively you can use Oligonucleotide 2 – a double-hairpin single labeled with fluorescein (for detection of a single type of DNA breaks (DNase II-type only). The single-fluorophore-carrying probe is considerably less expensive and is convenient whenever a single type of DNA break is to be labeled: 5′-AAG GGA CCT GCF GCA GGT CCC TTA ACG CAT ATG CGT T-3′; F – FITC-dT
- Oligonucleotide 3. Blunt-ended rhodamine-labeled hairpin for enhancement of in situ ligation signal with T4 DNA ligase: 5′-GCG CTA GAC CRG GTC TAG CGC-3′; R – Tetramethylrhodamine-dT
- Vaccinia DNA topoisomerase l (VACC TOPO) – 3000 U/μL (Vivid Technologies). In the initial experiments we used 215 pmoles (7.1 μg) of the VACC TOPO per every 25 μL of the reaction mix. However, the topoisomerase concentration can be significantly reduced without the loss of sensitivity. We later used a four times lesser amount of VACC TOPO per section (1.76 μg in 25 μL of the reaction mix per section) with similar results. Reducing amount of the enzyme to 880 ng (in 25 μL of the reaction mix) resulted in a weaker signal and 8.8 ng of VACC TOPO produced no detectable signal.
- T4 DNA ligase 5 U/μL (Roche). This highly concentrated ligase preparation gives the best signal in our experimental conditions.
- 10 x reaction buffer for T4 DNA ligase: 660 mM Tris-HCl, 50 mM MgCl2, 10 mM dithioerythritol, 10 mM ATP, pH 7.5 (20° C) (Roche) . ATP in reaction buffer is easily destroyed in repetitive cycles of thawing-freezing. Aliquot the buffer in small 15-20 μL portions and store at – 20° C. Use once.
- 30% (w/v) solution of PEG-8000 (Sigma) in bidistilled water. 15 % PEG-8000 in the reaction mix strongly stimulates the detection, increasing the effective concentrations of its constituents by volume exclusion.
- Proteinase K (Roche) 20 mg/mL stock solution in distilled water. Store at – 20° C. In the reaction use 50mg/mL solution in PBS, prepared from the stock. Do not reuse.
- Vectashield with DAPI (Vector Laboratories).
- Sodium bicarbonate buffer: 50mM NaHCO3, 15mM NaCl, pH 8.2.
- 22x22mm or 22x40mm glass or plastic coverslips. Plastic coverslips are preferable during the reaction, as they are easier to remove from the section.
References
- Meacham, G. C., Patterson, C., Zhang, W., Younger, J. M., Cyr, D. M. . Nature. Cell. Biol. 3, 100-105 (2001).
- Schuldt, A. Dynamic Pol expeditions. Nature. Cell. Biol. 4, E279-E279 (2002).
- Urry, D. W. Molecular machines: how motion and other functions of living organisms can result from reversible chemical changes. Angew. Chem. Int. Ed. Engl. 32, 819-841 (1993).
- Schneider, T. D. Theory of molecular machines I. Channel capacity of molecular machines. J. Theor. Biol. 148, 83-123 (1991).
- McClare, C. W. Chemical machines, Maxwell’s demon and living organisms. J. Theor. Biol. 30, 1-34 (1971).
- Spirin, A. S. Ribosome as a molecular machine. FEBS Lett. 514, 2-10 (2002).
- Fabbrizzi, L., Foti, F., Licchelli, M., Maccarini, P. M., Sacchi, D., Zema, M. Light-emitting molecular machines: pH-induced intramolecular motions in a fluorescent nickel(II) scorpionate complex. Chemistry. 8, 4965-4972 (2002).
- Drexler, K. E. . Nanosystems: Molecular Machinery, Manufacturing and Computation. , (1992).
- Freitas, R. A. . Nanomedicine: basic capabilities. , (1999).
- Didenko, V. V. DNA probes using fluorescence resonance energy transfer (FRET): designs and applications. BioTechniques. 31, 1106-1121 (2001).
- Minchew, C. L., Didenko, V. V. Fluorescent probes detecting the phagocytic phase of apoptosis: enzyme-substrate complexes of topoisomerase and DNA. Molecules. 16, 4599-4614 (2011).
- Didenko, V. V., Minchew, C. L., Shuman, S., Baskin, D. S. Semi-artificial fluorescent molecular machine for DNA damage detection. Nano. Letters. 4, 2461-2466 (2004).
- Didenko, V. V., Hornsby, P. J. Presence of double-strand breaks with single-base 3′ overhangs in cells undergoing apoptosis but not necrosis. J. Cell Biol. 135, 1369-1376 (1996).
- Didenko, V. V., Tunstead, J. R., Hornsby, P. J. Biotin-labeled hairpin oligonucleotides. Probes to detect double-strand breaks in DNA in apoptotic cells. Am. J. Pathol. 152, 897-902 (1998).
- Jones, R. A. L. . Soft Machines: Nanotechnology and life. , (2007).
- Samejima, K., Earnshaw, W. C. Trashing the genome: Role of nucleases during apoptosis. Nat. Rev. Mol. Cell. Biol. 6, 677-688 (2005).
