Name: dna-tethered rna polymerase for programmable in vitro transcription and molecular computation
Uploaded: 2021-12-29T13:00:00
Description: Watch this Scientific Journal Video about DNA-Tethered RNA Polymerase for Programmable In vitro Transcription and Molecular Computation at JoVE.com

L.Y.T.C感谢来自研究探索新前沿（NFRF-E），加拿大自然科学与工程研究委员会（NSERC）发现补助金以及多伦多大学医学设计倡议的慷慨支持，该倡议获得加拿大第一研究卓越基金（CFREF）的资助。

1. 缓冲液制备
注意：蛋白质纯化缓冲液的制备可以在任何一天进行;在这里，它是在开始实验之前完成的。
<ol><li>制备含有50mM三（羟甲基）氨基甲烷（Tris），300mM氯化钠（NaCl），5%甘油和5mM β巯基乙醇（BME）的裂解/平衡缓冲液，pH 8。在50 mL离心管中加入1.5 mL 1M Tris，1.8 mL 5M NaCl，1.5 mL甘油，25.2mL去离子水（ddH 2 O），并在使用前加入10.5μL14.2 M BME。 注意：Tris可引起?...

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	<li>Cherry, K. M., Qian, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scaling+up+molecular+pattern+recognition+with+DNA-based+winner-take-all+neural+networks.">Scaling up molecular pattern recognition with DNA-based winner-take-all neural networks.</a> Nature. 559 (7714), 370-376 (2018).</li><li>Qian, L., Winfree, E., Bruck, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+network+computation+with+DNA+strand+displacement+cascades.">Neural network computation with DNA strand displacement cascades.</a> Nature. 475 (7356), 368-372 (2011).</li><li>Chen, Y. -. J., 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=Programmable+chemical+controllers+made+from+DNA.">Programmable chemical controllers made from DNA.</a> Nature Nanotechnology. 8 (10), 755-762 (2013).</li><li>di Bernardo, D., Marucci, L., Menolascina, F., Siciliano, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predicting+synthetic+gene+networks.">Predicting synthetic gene networks.</a> Synthetic Gene Networks: Methods and Protocols. 813, 57-81 (2012).</li><li>Xiang, Y., Dalchau, N., Wang, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scaling+up+genetic+circuit+design+for+cellular+computing:+advances+and+prospects.">Scaling up genetic circuit design for cellular computing: advances and prospects.</a> Natural Computing. 17 (4), 833-853 (2018).</li><li>Gould, N., Hendy, O., Papamichail, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computational+tools+and+algorithms+for+designing+customized+synthetic+genes.">Computational tools and algorithms for designing customized synthetic genes.</a> Frontiers in Bioengineering and Biotechnology. 2, (2014).</li><li>MacDonald, J. T., Siciliano, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computational+sequence+design+with+R2oDNA+Designer.">Computational sequence design with R2oDNA Designer.</a> Mammalian Synthetic Promoters. 1651, 249-262 (2017).</li><li>Cervantes-Salido, V. M., Jaime, O., Brizuela, C. A., Mart&#237;nez-P&#233;rez, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+the+design+of+sequences+for+DNA+computing:+A+multiobjective+evolutionary+approach.">Improving the design of sequences for DNA computing: A multiobjective evolutionary approach.</a> Applied Soft Computing. 13 (12), 4594-4607 (2013).</li><li>Zadeh, J. N., 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=NUPACK:+Analysis+and+design+of+nucleic+acid+systems.">NUPACK: Analysis and design of nucleic acid systems.</a> Journal of Computational Chemistry. 32 (1), 170-173 (2011).</li><li>Fornace, M. E., Porubsky, N. J., Pierce, N. 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+unified+dynamic+programming+framework+for+the+analysis+of+interacting+nucleic+acid+strands:+enhanced+models,+scalability,+and+speed.">A unified dynamic programming framework for the analysis of interacting nucleic acid strands: enhanced models, scalability, and speed.</a> ACS Synthetic Biology. 9 (10), 2665-2678 (2020).</li><li>Wetterstrand, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+sequencing+costs:+Data.">DNA sequencing costs: Data.</a> Genome.gov. , (2020).</li><li>Lopez, R., Wang, R., Seelig, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+molecular+multi-gene+classifier+for+disease+diagnostics.">A molecular multi-gene classifier for disease diagnostics.</a> Nature Chemistry. 10 (7), 746-754 (2018).</li><li>Pardee, K., 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=low-cost+detection+of+Zika+virus+using+programmable+biomolecular+components.">low-cost detection of Zika virus using programmable biomolecular components.</a> Cell. 165 (5), 1255-1266 (2016).</li><li>Yurke, B., Turberfield, A. J., Mills, A. P., Simmel, F. C., Neumann, J. 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+DNA-fuelled+molecular+machine+made+of+DNA.">A DNA-fuelled molecular machine made of DNA.</a> Nature. 406 (6796), 605-608 (2000).</li><li>Lin, K. N., Volkel, K., Tuck, J. M., Keung, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+and+scalable+DNA-based+information+storage.">Dynamic and scalable DNA-based information storage.</a> Nature Communications. 11 (1), 2981 (2020).</li><li>Yurke, B., Mills, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+DNA+to+power+nanostructures.">Using DNA to power nanostructures.</a> Genetic Programming and Evolvable Machines. 4 (2), 111-122 (2003).</li><li>Zhang, D. Y., Turberfield, A. J., Yurke, B., Winfree, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+entropy-driven+reactions+and+networks+catalyzed+by+DNA.">Engineering entropy-driven reactions and networks catalyzed by DNA.</a> Science. 318 (5853), 1121-1125 (2007).</li><li>Wang, B., Thachuk, C., Ellington, A. D., Winfree, E., Soloveichik, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+design+principles+for+leakless+strand+displacement+systems.">Effective design principles for leakless strand displacement systems.</a> Proceedings of the National Academy of Sciences. 115 (52), 12182-12191 (2018).</li><li>Machinek, R. R. F., Ouldridge, T. E., Haley, N. E. C., Bath, J., Turberfield, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Programmable+energy+landscapes+for+kinetic+control+of+DNA+strand+displacement.">Programmable energy landscapes for kinetic control of DNA strand displacement.</a> Nature Communications. 5 (1), 5324 (2014).</li><li>Cabello-Garcia, J., Bae, W., Stan, G. -. B. V., Ouldridge, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Handhold-mediated+strand+displacement:+a+nucleic+acid-based+mechanism+for+generating+far-from-equilibrium+assemblies+through+templated+reactions.">Handhold-mediated strand displacement: a nucleic acid-based mechanism for generating far-from-equilibrium assemblies through templated reactions.</a> bioRxiv. , (2020).</li><li>Brophy, J. A. N., Voigt, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+genetic+circuit+design.">Principles of genetic circuit design.</a> Nature Methods. 11 (5), 508-520 (2014).</li><li>Khalil, A. 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=A+synthetic+biology+framework+for+programming+eukaryotic+transcription+functions.">A synthetic biology framework for programming eukaryotic transcription functions.</a> Cell. 150 (3), 647-658 (2012).</li><li>Swank, Z., Laohakunakorn, N., Maerkl, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+gene-regulatory+network+engineering+with+synthetic+transcription+factors.">Cell-free gene-regulatory network engineering with synthetic transcription factors.</a> Proceedings of the National Academy of Sciences. 116 (13), 5892-5901 (2019).</li><li>Howland, S. W., Tsuji, T., Gnjatic, S., Ritter, G., Old, L. J., Wittrup, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducing+efficient+cross-priming+using+antigen-coated+yeast+particles.">Inducing efficient cross-priming using antigen-coated yeast particles.</a> Journal of immunotherapy. 31 (7), 607 (2008).</li><li>Abil, Z., Ellefson, J. W., Gollihar, J. D., Watkins, E., Ellington, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compartmentalized+partnered+replication+for+the+directed+evolution+of+genetic+parts+and+circuits.">Compartmentalized partnered replication for the directed evolution of genetic parts and circuits.</a> Nature Protocols. 12 (12), 2493-2512 (2017).</li><li>Baugh, C., Grate, D., Wilson, C., Doudna, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2.8+&#197;+crystal+structure+of+the+malachite+green+aptamer11.">2.8 &#197; crystal structure of the malachite green aptamer11.</a> Journal of Molecular Biology. 301 (1), 117-128 (2000).</li><li>Chou, L. Y. T., Shih, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+transcriptional+regulation+via+nucleic+acid-based+transcription+factors.">In vitro transcriptional regulation via nucleic acid-based transcription factors.</a> ACS Synthetic Biology. 8 (11), 2558-2565 (2019).</li><li>Lykke-Andersen, J., Christiansen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+C-terminal+carboxy+group+of+T7+RNA+polymerase+ensures+efficient+magnesium+ion-dependent+catalysis.">The C-terminal carboxy group of T7 RNA polymerase ensures efficient magnesium ion-dependent catalysis.</a> Nucleic Acids Research. 26 (24), 5630-5635 (1998).</li><li>Pu, J., Disare, M., Dickinson, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+C-terminal+modification+tolerance+in+full-length+and+split+T7+RNA+Polymerase+biosensors.">Evolution of C-terminal modification tolerance in full-length and split T7 RNA Polymerase biosensors.</a> Chembiochem. 20 (12), 1547-1553 (2019).</li><li>Gardner, L. P., Mookhtiar, K. A., Coleman, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initiation,+elongation,+and+processivity+of+carboxyl-terminal+mutants+of+T7+RNA+polymerase.">Initiation, elongation, and processivity of carboxyl-terminal mutants of T7 RNA polymerase.</a> Biochemistry. 36 (10), 2908-2918 (1997).</li><li>Yin, J., Lin, A. J., Golan, D. E., Walsh, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-specific+protein+labeling+by+Sfp+phosphopantetheinyl+transferase.">Site-specific protein labeling by Sfp phosphopantetheinyl transferase.</a> Nature Protocols. 1 (1), 280-285 (2006).</li><li>Warden-Rothman, R., Caturegli, I., Popik, V., Tsourkas, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sortase-tag+expressed+protein+ligation:+combining+protein+purification+and+site-specific+bioconjugation+into+a+single+step.">Sortase-tag expressed protein ligation: combining protein purification and site-specific bioconjugation into a single step.</a> Analytical Chemistry. 85 (22), 11090-11097 (2013).</li><li>Zhang, W. -. B., Sun, F., Tirrell, D. A., Arnold, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+macromolecular+topology+with+genetically+encoded+SpyTag-SpyCatcher+chemistry.">Controlling macromolecular topology with genetically encoded SpyTag-SpyCatcher chemistry.</a> Journal of the American Chemical Society. 135 (37), 13988-13997 (2013).</li></ol>

本研究展示了一种受DNA纳米技术启发的方法，通过N末端SNAP标记的重组T7 RNAP与BG功能化的寡核苷酸共价耦合来控制T7 RNA聚合酶的活性，随后用于编程TMDSD反应。根据设计，SNAP标签位于聚合酶的N端，因为野生型T7 RNAP的C端埋在蛋白质结构核心内并与DNA模板28进行重要接触。先前修改聚合酶C末端的尝试已导致酶活性完全丧失，除非引入其他补偿性突变。29，30</sup...

DNA计算使用一组设计的寡核苷酸作为计算介质。这些寡核苷酸被编程为序列，以根据用户指定的逻辑动态组装并响应特定的核酸输入。在概念验证研究中，计算的输出通常由一组荧光标记的寡核苷酸组成，可以通过凝胶电泳或荧光板读数器进行检测。在过去的30年中，已经证明了越来越复杂的DNA计算电路，例如各种数字逻辑级联，化学反应网络和神经网络1，2，3。为了帮助制备这些DNA回路，数学模型已被用于预测合成基因回路4，5的功能，并且已经开发了用于正交DNA序列设计6，7，8，9，10的计算工具。.与硅基计算机相比，DNA计算机的优势包括它们能够直接与生物分子连接，在没有电源的情况下在溶液中运行，以及它们的整体紧凑性和稳定性。随着下一代测序的出现，合成DNA计算机的成本在过去二十年中一直在以比摩尔定律11更快的速度下降。这种基于DNA的计算机的应用现在开始出现，例如用于疾病诊断12，13，用于为分子生物物理学提供动力14，以及作为数据存储平台15。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62073/62073fig01.jpg"> 图1：脚趾介导的DNA链位移的机制。 δ，脚趾是部分双工上的自由、未绑定序列。当在第二条链上引入互补域（δ*）时，自由δ域充当杂交的支架，允许链的其余部分（ɑ*）通过称为链迁移的压缩/解压缩可逆反应缓慢地取代其竞争对手。随着δ长度的增加，正向反应的ΔG减小，位移更容易发生。 <a href="https://www.jove.com/files/ftp_upload/62073/62073fig01large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>
迄今为止，大多数DNA计算机都利用动态DNA纳米技术领域公认的基序，称为脚趾介导的DNA链置换（TMDSD，图1）16。该基序由部分双链DNA（dsDNA）双链组成，显示短的"脚趾"悬垂（即7至10个核苷酸（nt））。核酸"输入"链可以通过脚趾与部分双链相互作用。这导致其中一条链从部分双工移出，然后这个释放的链可以作为下游部分双工的输入。因此，TMDSD可实现信号级联和信息处理。原则上，正交TMDSD基序可以在解决方案中独立运行，从而实现并行信息处理。TMDSD反应存在许多变化，例如脚趾介导的DNA链交换（TMDSE）17，具有双长结构域18的"无泄漏"脚趾，序列不匹配的脚趾19和"手持"介导的链位移20。这些创新的设计原则允许更精细地调整TMDSD能量学和动力学，以提高DNA计算性能。
合成基因回路，如转录基因回路，也能够计算21、22、23。这些回路由蛋白质转录因子调节，蛋白质转录因子通过与特定的调节DNA元件结合来激活或抑制基因的转录。与基于DNA的电路相比，转录电路具有几个优点。首先，酶转录比现有的催化DNA回路具有更高的周转率，因此每个输入拷贝产生更多的输出拷贝，并提供更有效的信号放大手段。此外，转录电路可以产生不同的功能分子，如适配子或编码治疗蛋白的信使RNA（mRNA）作为计算输出，可以用于不同的应用。然而，当前转录电路的一个主要限制是它们缺乏可扩展性。这是因为基于正交蛋白质的转录因子集非常有限，并且新蛋白质转录因子的从头设计在技术上仍然具有挑战性且耗时。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62073/62073fig02.jpg"> 图2："系绳"和"笼式"聚合酶复合物的提取和机理（A和B）寡核苷酸系绳通过SNAP标签反应酶被酶标记为T7聚合酶。由具有系绳补体悬垂的"人造"T7启动子组成的笼子使其能够与系绳杂交并阻断转录活性。（C）当操作者（a*b*）存在时，它结合到寡核苷酸系绳（ab）上的脚趾并取代笼子的b*区域，从而允许转录发生。这个数字是从Chou和Shih27修改而来的。缩写：RNAP = RNA 聚合酶。<a href="https://www.jove.com/files/ftp_upload/62073/62073fig02large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>
本文介绍了一种用于分子计算的新型构建块，它将转录电路的功能与基于DNA的电路的可扩展性相结合。该构建块是与单链DNA系绳共价连接的T7RNAP（图2A）。为了合成这种DNA系留的T7 RNAP，将聚合酶融合到N端SNAP标签24中并在大肠杆菌中重组表达。然后将SNAP标签与与BG底物功能化的寡核苷酸反应。寡核苷酸系绳允许分子来宾通过DNA杂交定位在靠近聚合酶的位置。其中一位客人是一种被称为"笼子"的竞争性转录阻滞剂，它由一个"人造"T7启动子DNA双链体组成，下游没有基因（图2B）。当通过其寡核苷酸系绳与RNAP结合时，笼子通过与其他DNA模板竞争RNAP结合来阻止聚合酶活性，从而使RNAP处于"OFF"状态（图2C）。
为了将聚合酶激活到"ON"状态，设计了具有基因T7启动子上游的单链"操作员"结构域的T7 DNA模板。操作结构域（即结构域a*b*图2C）可以设计为通过TMDSD将笼子从RNAP取代，并将RNAP定位在基因T7启动子的近端，从而启动转录。或者，还设计了DNA模板，其中操作序列与称为"人工转录因子"的辅助核酸链（即图3A中的TFA和TFB链）互补。当两条链都被引入反应中时，它们将在操作员现场组装，从而创建一个新的伪连续域a*b*。然后，该结构域可以通过TMDSD取代笼子以启动转录（图3B）。这些链可以外源供应或生产。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62073/62073fig03.jpg"> 图3：通过三组分开关激活剂对聚合酶活性进行选择性编程。 （A）当转录因子（TFA 和TFB）存在时，它们与启动子上游的操作员结构域结合，形成一个伪单链序列（a*b*），能够通过脚趾介导的DNA置换来置换笼子。（B） 这个 a*b* 结构域可以通过 TMDSD 取代笼子以启动转录。这个数字是从Chou和Shih27修改而来的。缩写： TF = 转录因子;RNAP = RNA聚合酶;TMDSD = 脚趾介导的 DNA 链置换。 <a href="https://www.jove.com/files/ftp_upload/62073/62073fig03large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>
使用基于核酸的转录因子进行体外转录调控允许可扩展地实现复杂的电路行为，如数字逻辑、反馈和信号级联。例如，人们可以通过设计核酸序列来构建逻辑门级联，使得来自上游基因的转录本激活下游基因。利用该技术所提出的级联和多路复用能力的一个应用是开发更复杂的分子计算电路，用于便携式诊断和分子数据处理。此外，集成分子计算和从头RNA合成功能可以实现新的应用。例如，分子电路可以设计用于检测用户定义的RNA的一种或组合，作为输入和输出治疗性RNA或mRNA编码功能肽或蛋白质的mRNA，用于床旁医疗应用。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.5% polysorbate 20 (TWEEN 20)</td>
			<td>BioShop</td>
			<td>TWN510.5</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5M ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Bio Basic</td>
			<td>SD8135</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mM sodium phosphate buffer (pH 7)</td>
			<td>Bio Basic</td>
			<td>PD0435</td>
			<td>Tablets used to make 10 mM buffer</td>
		</tr>
		<tr>
			<td>10% ammonium persulfate (APS)</td>
			<td>Sigma Aldrich</td>
			<td>A3678-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>100 kDa Amicon Ultra-15 Centrifugal Filter Unit</td>
			<td>Fisher Scientific</td>
			<td>UFC910008</td>
			<td></td>
		</tr>
		<tr>
			<td>100% acetone</td>
			<td>Fisher Chemical</td>
			<td>A18P4</td>
			<td></td>
		</tr>
		<tr>
			<td>100% ethanol (EtOH)</td>
			<td>House Brand</td>
			<td>39752-P016-EAAN</td>
			<td></td>
		</tr>
		<tr>
			<td>10x in vitro transcription (IVT) buffer</td>
			<td>New England Biolabs</td>
			<td>B9012</td>
			<td></td>
		</tr>
		<tr>
			<td>10x Tris-Borate-EDTA (TBE) buffer</td>
			<td>Bio Basic</td>
			<td>A0026</td>
			<td></td>
		</tr>
		<tr>
			<td>1M Isopropyl &#946;- d-1-thiogalactopyranoside (IPTG)</td>
			<td>Sigma Aldrich</td>
			<td>I5502-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>1M sodium bicarbonate buffer</td>
			<td>Sigma Aldrich</td>
			<td>S6014-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>1M Tris(hydroxymethyl)aminomethane (Tris)</td>
			<td>Sigma Aldrich</td>
			<td>648311-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>1X Tris-EDTA (TE) buffer</td>
			<td>ThermoFisher</td>
			<td>12090015</td>
			<td></td>
		</tr>
		<tr>
			<td>2M imidazole</td>
			<td>Sigma Aldrich</td>
			<td>56750-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>2-mercaptoethanol (BME)</td>
			<td>Sigma Aldrich</td>
			<td>M3148</td>
			<td></td>
		</tr>
		<tr>
			<td>3M sodium acetate</td>
			<td>Bio Basic</td>
			<td>SRB1611</td>
			<td></td>
		</tr>
		<tr>
			<td>40% acrylamide (19:1)</td>
			<td>Bio Basic</td>
			<td>A00062</td>
			<td></td>
		</tr>
		<tr>
			<td>4x LDS protein sample loading buffer</td>
			<td>Fisher Scientific</td>
			<td>NP0007</td>
			<td></td>
		</tr>
		<tr>
			<td>5M sodium chloride (NaCl)</td>
			<td>Bio Basic</td>
			<td>DB0483</td>
			<td></td>
		</tr>
		<tr>
			<td>5mM dithiothreitol (DTT)</td>
			<td>Sigma Aldrich</td>
			<td>43815-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>6x gel loading dye</td>
			<td>New England Biolabs</td>
			<td>B7024S</td>
			<td></td>
		</tr>
		<tr>
			<td>agarose B powder</td>
			<td>Bio Basic</td>
			<td>AB0014</td>
			<td></td>
		</tr>
		<tr>
			<td>BG-GLA-NHS</td>
			<td>New England Biolabs</td>
			<td>S9151S</td>
			<td></td>
		</tr>
		<tr>
			<td>BL21 competent E. coli</td>
			<td>Addgene</td>
			<td>C2530H</td>
			<td></td>
		</tr>
		<tr>
			<td>BLUeye prestained protein ladder</td>
			<td>FroggaBio</td>
			<td>PM007-0500</td>
			<td></td>
		</tr>
		<tr>
			<td>bromophenol blue</td>
			<td>Bio Basic</td>
			<td>BDB0001</td>
			<td></td>
		</tr>
		<tr>
			<td>coomassie blue (SimplyBlue SafeStain)</td>
			<td>ThermoFisher</td>
			<td>LC6060</td>
			<td></td>
		</tr>
		<tr>
			<td>cyanine dye (SYBR Gold nucleic acid gel stain)</td>
			<td>Fisher Scientific</td>
			<td>S11494</td>
			<td></td>
		</tr>
		<tr>
			<td>cyanine dye (SYBR Safe nucleic acid gel stain)</td>
			<td>Fisher Scientific</td>
			<td>S33102</td>
			<td></td>
		</tr>
		<tr>
			<td>dry dimethyl sulfoxide (DMSO)</td>
			<td>Fisher Scientific</td>
			<td>D12345</td>
			<td></td>
		</tr>
		<tr>
			<td>formamide</td>
			<td>Sigma Aldrich</td>
			<td>F9037-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>glycerol</td>
			<td>Bio Basic</td>
			<td>GB0232</td>
			<td></td>
		</tr>
		<tr>
			<td>kanamycin sulfate</td>
			<td>BioShop</td>
			<td>KAN201.5</td>
			<td></td>
		</tr>
		<tr>
			<td>lysogeny broth</td>
			<td>Sigma Aldrich</td>
			<td>L2542-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>malachite green oxalate</td>
			<td>Sigma Aldrich</td>
			<td>2437-29-8</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N,N&#39;N&#39;-Tetramethylethane-1,2-diamine (TEMED)</td>
			<td>Sigma Aldrich</td>
			<td>T9281-25ML</td>
			<td></td>
		</tr>
		<tr>
			<td>NuPAGE MES SDS running buffer (20x)</td>
			<td>Fisher Scientific</td>
			<td>LSNP0002</td>
			<td></td>
		</tr>
		<tr>
			<td>NuPAGE Novex 4-12% Bis-Tris gel 1.0 mm 12-well</td>
			<td>Life Technologies</td>
			<td>NP0322BOX</td>
			<td></td>
		</tr>
		<tr>
			<td>oligonucleotide (cage antisense)</td>
			<td>IDT</td>
			<td>N/A</td>
			<td>TATAGTGAGTCGTATTAATTTG</td>
		</tr>
		<tr>
			<td>oligonucleotide (cage sense)</td>
			<td>IDT</td>
			<td>N/A</td>
			<td>TCAGTCACCTATCTGTTTCAAA 
			TTAATACGACTCACTATA</td>
		</tr>
		<tr>
			<td>oligonucleotide (malachite green aptamer antisense)</td>
			<td>IDT</td>
			<td>N/A</td>
			<td>GGATCCATTCGTTACCTGGCT 
			CTCGCCAGTCGGGATCCTATA 
			GTGAGTCGTATTACAGTTCCAT 
			TATCGCCGTAGTTGGTGTACT</td>
		</tr>
		<tr>
			<td>oligonucleotide (malachite green aptamer sense)</td>
			<td>IDT</td>
			<td>N/A</td>
			<td>TAATACGACTCACTATAGGATC 
			CCGACTGGCGAGAGCCAGGT 
			AACGAATGGATCC</td>
		</tr>
		<tr>
			<td>oligonucleotide (Transcription Factor A)</td>
			<td>IDT</td>
			<td>N/A</td>
			<td>AGTACACCAACTACGAGTGAG</td>
		</tr>
		<tr>
			<td>oligonucleotide (Transcription Factor B)</td>
			<td>IDT</td>
			<td>N/A</td>
			<td>TCAGTCACCTATCTGGCGATAA 
			TGGAACTG</td>
		</tr>
		<tr>
			<td>oligonucleotide with 3&#8217; Amine modification (tether)</td>
			<td>IDT</td>
			<td>N/A</td>
			<td>GCTACTCACTCAGATAGGTGAC 
			TGA/3AmMO/</td>
		</tr>
		<tr>
			<td>Pierce strong ion exchange spin columns</td>
			<td>Fisher Scientific</td>
			<td>90008</td>
			<td></td>
		</tr>
		<tr>
			<td>plasmid encoding SNAP T7 RNAP and kanamycin resistance genes</td>
			<td>Genscript</td>
			<td>N/A</td>
			<td>custom gene insert</td>
		</tr>
		<tr>
			<td>protein purification column (HisPur Ni-NTA spin column)</td>
			<td>Fisher Scientific</td>
			<td>88226</td>
			<td></td>
		</tr>
		<tr>
			<td>rNTP mix</td>
			<td>New England Biolabs</td>
			<td>N0466S</td>
			<td></td>
		</tr>
		<tr>
			<td>Roche mini quick DNA spin column</td>
			<td>Sigma Aldrich</td>
			<td>11814419001</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma Aldrich</td>
			<td>T8787-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra Low Range DNA ladder</td>
			<td>Fisher Scientific</td>
			<td>10597012</td>
			<td></td>
		</tr>
		<tr>
			<td>urea</td>
			<td>BioShop</td>
			<td>URE001.1</td>
			<td></td>
		</tr>
	</tbody>
</table>

dna-tethered rna polymerase for programmable in vitro transcription and molecular computation

DNA纳米技术使核酸的可编程自组装成用户规定的形状和动态，适用于各种应用。这项工作表明，DNA纳米技术的概念可用于编程噬菌体衍生的T7 RNA聚合酶（RNAP）的酶活性，并构建可扩展的合成基因调节网络。首先，通过表达N末端SNAP标记RNAP以及随后SNAP标签与苄基胍宁（BG）修饰的寡核苷酸的化学偶联来设计寡核苷酸系留的T7 RNAP。接下来，核酸链置换用于按需编程聚合酶转录。此外，辅助核酸组装体可用作"人工转录因子"，以调节DNA程序化的T7 RNAP与其DNA模板之间的相互作用。这种体外转录调节机制可以实现各种电路行为，如数字逻辑、反馈、级联和多路复用。这种基因调控架构的可组合性促进了设计抽象、标准化和缩放。这些功能将使体外遗传设备的快速原型设计成为可能，用于生物传感、疾病检测和数据存储等应用。

<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62073/62073fig05.jpg"> 图5：SNAP T7 RNAP表达和体外转录测定的SDS-PAGE分析。（A）SNAP T7 RNAP蛋白纯化分析，SNAP T7 RNAP分子量：119.4kDa。FT=从柱中流出，W1=含有杂质的洗涤缓冲液的洗脱级分，E1-3=含有纯化产物的洗脱级分，并且DE=10倍稀释的脱盐总洗脱。4-12%预制双-Tris蛋白凝胶，染色：考马...

Watch this Scientific Journal Video about DNA-Tethered RNA Polymerase for Programmable In vitro Transcription and Molecular Computation at JoVE.com

DNA-Tethered RNA Polymerase for Programmable In vitro Transcription and Molecular Computation

我们描述了一种新型DNA系留T7 RNA聚合酶的工程设计，以调节体外转录反应。我们讨论了蛋白质合成和表征的步骤，验证了概念验证转录调控，并讨论了其在分子计算、诊断和分子信息处理中的应用。

DNA系留RNA聚合酶用于可编程体外转录和分子计算

DNA nanotechnology enables programmable self-assembly of nucleic acids into user-prescribed shapes and dynamics for diverse applications. This work ...

我们提出了一种使用人工核酸转录因子调节聚合酶活性的方法。这种基因调控架构可以作为未来体外遗传装置的基石。通过将DNA结合域与T7 RNA聚合酶相关联，该技术将基于DNA的电路的可扩展性与转录电路的功能相结合。首先将9个稀释的单链BG寡核苷酸稀释到最终体积为50微升的双蒸馏水，比例从5到1到1到5，如表中所示。通过将两微升SNAP缓冲液与四微升BG寡核苷酸和四微升SNAP T7 RNAP混合，为每个稀释液制备一个反应混合物。通过用双蒸馏水代替寡核苷酸来制备RNAP对照，并通过用双蒸馏水代替SNAP T7 RNAP来制备DNA对照。通过将每个样品的2微升加入4微升的SNAP缓冲液和每个样品的2微升蛋白质上样染料来设置11个反应。在70摄氏度下加热反应10分钟，然后将每个样品的两微升加载到4至12%的玻璃体蛋白质凝胶上，在200伏的冰上进行35分钟的凝胶电泳。在运行结束时，用三次水交换在摇床上洗涤凝胶，每次洗涤至少10分钟，然后用菁染料进行核酸染色15分钟。在适当的凝胶成像系统上对凝胶进行成像，并用20毫升考马斯蓝再次染色凝胶。一小时后，用双蒸馏水去污至少一小时，然后再对凝胶进行成像。对于寡核苷酸系留的SNAP T7纯化，首先，通过将50微升一摩尔痕量，与100微升五摩尔氯化钠，与850微升双蒸馏水混合，制备洗脱缓冲液。接下来，将每个样品的一个离子交换柱放入单独的两毫升离心管中，并通过离心用纯化缓冲液洗涤色谱柱，直到所有缓冲液都从每个色谱柱中洗脱出来。用纯化缓冲液以三比一纯化缓冲液与样品的比例稀释每个样品，并将400微升样品倒入每个色谱柱中。当所有缓冲液都已洗脱后，如图所示，收集并标记每个样品的流经。每次洗涤时用400微升纯化缓冲液洗涤色谱柱三次，直到每次洗涤所有缓冲液都洗脱，并根据需要收集和标记流经物。最后一次洗涤后，通过将50微升洗脱缓冲液加入柱中心并离心柱三次直到所有50微升缓冲液洗脱出来，收集洗脱的蛋白质。每次离心后收集并标记洗脱液，在最后一次离心后将洗脱液池入单个管中，同时留下少量每个洗脱用于凝胶分析。在260和280纳米处测量剩余洗脱等分试样的吸光度，并以一比一的比例向样品中加入甘油，以储存零下20度，直到使用。使用500微升30千尔顿离心过滤单元缓冲液，用2x储存缓冲液交换总洗脱产物。并测量吸光度，如图所示。然后以一比一的比例向产品中加入甘油，以储存零下20摄氏度。要通过SDS-PAGE评估洗脱，请在4%至12%的玻璃体凝胶上以适当的蛋白质分子量标准品在200伏电压下运行样品35分钟，或直到染料前部迁移到凝胶末端。为了证明系留RNAP的按需控制活性，将每个模板的2.4微升与5微升5x退火缓冲液和14.2微升双蒸馏水混合。将得到的一个微摩尔双链DNA CAGE在75摄氏度下孵育两分钟。并以与表中所示相同的方式退火启动子和孔雀石绿色适配体DNA模板的感官和反感官菌株。在孵育结束时，将系留的SNAP T7 RNAP以1至5摩尔比加入双链DNA CAGE中，最终浓度为500纳摩尔RNAP。在室温下15分钟后，将样品放在冰上，并将2.5微升10x体外转录缓冲液与1微升25毫摩尔核糖核苷三磷酸盐，1微升1毫摩尔孔雀石绿，2.5微升RNAP CAGE混合物，2.5微升一个微摩尔转录因子A和B寡核苷酸链， 和三微升的一毫摩尔孔雀石绿色适配子模板在10微升双蒸馏水中。在15微升双蒸馏水中，建立含有2.5微升10x体外转录缓冲液，一微升25毫摩尔核糖核苷三磷酸混合物，一微升一微升1毫摩尔孔雀石绿，2.5微升RNAP CAGE混合物和三微升一毫摩尔孔雀石绿色适配子模板的第二反应。为了建立仅含有缓冲液的反应，在17.5微升双蒸馏水中混合2.5微升10x体外转录缓冲液，一微升25毫摩尔核糖苷三磷酸混合物，一微升一毫摩尔孔雀石绿和三微升一毫摩尔孔雀石绿色适配体模板。当所有反应都准备好后，将每个反应转移到384孔板中，并在荧光板读数仪上监测孔雀石绿色适配子转录两小时，在37摄氏度下以610纳米激发和655纳米发射。在读数结束时，将板放在冰上，并将RNA产物在变性12%TBE聚丙烯酸凝胶中，在70摄氏度TBE缓冲液中以280伏特运行20分钟，或直到染料前端到达凝胶的末端。然后，在眼眶振荡器上用菁染料核酸染色凝胶10分钟，然后成像，如图所示。重组 SNAP T7 RNAP 蛋白的成功表达和纯化可以使用 SDS-PAGE 进行确认。在SDS-PAGE之后，酶活性可以通过体外转录反应来验证。BG官能化寡核苷酸的成功偶联可以通过18%变性TBE-尿素PAGE进行验证。与未修饰的寡核苷酸相比，将BG部分添加到寡核苷酸中会增加其分子量。正如在该具有代表性的菁染染料染色凝胶中观察到的，以确认从过量的BG寡核苷酸中纯化DNA系留的T7 RNAP，初始流经部分主要含有过量的BG寡核苷酸，以及一小部分未与阳离子交换树脂结合的DNA系留聚合酶。池稀释级分仅含有由菁染料与寡核苷酸系绳结合引起的RNAP寡核苷酸单条带，上浓缩产物泳道表现出相同但较暗的条带，表示成功的上调浓度。通过考马斯蓝染色观察到相同的模式，在非偶联RNAP对照中观察到小的凝胶迁移率变化。在这里，在体外转录结束时产生的荧光信号被实时动力学显示。在该分析中，观察到荧光信号中336倍的活化，证明了对聚合酶活性的稳健控制。尝试此方案时，请记住在核酸染色之前彻底洗涤凝胶上的SDS。这是因为STS会将染料捕获在凝胶中，从而导致高背景信号。将我们的方法应用于具有多个级联的大型基因回路将展示系留转录系统的可扩展性和可组合性。该技术目前正被用于开发能够进行神经网络样式计算的基因电路。

Research

JoVE Journal

Bioengineering

Adaptation of a Haptic Robot in a 3T fMRI

触觉机器人适应在3T功能磁共振成像

Functional magnetic resonance imaging (fMRI) provides excellent functional brain imaging via the BOLD signal 1 with advantages including non-ionizing ...

科研

生物工程

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

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

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 ...

The Encapsulation of Cell-free Transcription and Translation Machinery in Vesicles for the Construction of Cellular Mimics

无细胞的转录和翻译的机械囊泡的封装建设模拟蜂窝

As interest shifts from individual molecules to systems of molecules, an increasing number of laboratories have sought to build from the bottom up ...

Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

釉原蛋白 - 壳聚糖水凝胶的发展<em&gt;体外</em&gt;搪瓷愈合了密集接口

Biomimetic enamel reconstruction is a significant topic in material science and dentistry as a novel approach for the treatment of dental caries or ...

An In Vitro Enzymatic Assay to Measure Transcription Inhibition by Gallium(III) and H3 5,10,15-tris(pentafluorophenyl)corroles

An In Vitro Enzymatic Assay to Measure Transcription Inhibition by GalliumIII and H3 5,10,15-trispentafluorophenylcorroles

一个<em&gt;在体外</em&gt;酶测定来测量转录抑制作用镓（Ⅲ）和H<sub&gt; 3</sub&gt; -5,10,15-三（五氟苯基）corroles

Chemotherapy often involves broad-spectrum cytotoxic agents with many side effects and limited targeting. Corroles are a class of tetrapyrrolic ...

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 ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

一个<em&gt;体外</em鼠类的椎间盘&gt;器官培养模型

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

用化学表观修饰因子重定向细胞机械抑制基因转录

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene ...

Generation of Cationic Nanoliposomes for the Efficient Delivery of In Vitro Transcribed Messenger RNA

Generation of Cationic Nanoliposomes for the Efficient Delivery of In Vitro Transcribed Messenger RNA

阳离子纳米脂质体的生成, 用于高效输送体外转录信使 rna

The development of messenger RNA (mRNA)-based therapeutics for the treatment of various diseases becomes more and more important because of the positive ...

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 ...