Name: real-time imaging of single engineered rna transcripts in living cells using ratiometric bimolecular beacons
Uploaded: 2014-08-06T14:30:00
Description: Watch this Scientific Journal Video about Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons at JoVE.com

这项工作得到了美国国家科学基金会成就奖（0953583）和健康NCI / R21-CA116102的研究所，NCI / R21-CA125088，NIBIB / R01-EB012065，NCI / R01-CA157766支持。

在这个协议中，一个寡核苷酸（RBMB1）被标记在它的5&#39;末端与CF640R报告染料和具有序列：5&#39;-mCmUmUmC mGmUmC mCmAmC mAmAmA mCmAmC mAmAmC mUmCmC亩mGmAmAmG mGmAmC mGmGmC mAmGmC mGmUmG mCmAmG mCmUmC木木-3&#39;。自身互补结构域，其驱动所述发夹结构的形成中，用粗体表示。第二个寡核苷酸（RBMB2）被标记在3&#39;端有一个爱荷华黑色RQ-SP猝灭剂和具有序列：5&#39;-mGmAmG mCmUmG mCmAmC mGmCmU mGmCmC mGmUmC-3&#39...

<ol>
	<li>Tyagi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+intracellular+RNA+distribution+and+dynamics+in+living+cells.">Imaging intracellular RNA distribution and dynamics in living cells.</a> Nat Methods. 6 (5), 331-338 (2009).</li><li>Bertrand, E., Chartrand, P., Schaefer, M., Shenoy, S. M., Singer, R. H., Long, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+ASH1+mRNA+particles+in+living+yeast.">Localization of ASH1 mRNA particles in living yeast.</a> Mol Cell. 2 (4), 437-445 (1998).</li><li>Dictenberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+encoding+of+fluorescent+RNA+ensures+a+bright+future+for+visualizing+nucleic+acid+dynamics.">Genetic encoding of fluorescent RNA ensures a bright future for visualizing nucleic acid dynamics.</a> Trends Biotechnol. 30 (12), 621-626 (2012).</li><li>Chubb, J. R., Trcek, T., Shenoy, S. M., Singer, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+pulsing+of+a+developmental+gene.">Transcriptional pulsing of a developmental gene.</a> Curr Biol. 16 (10), 1018-1025 (2006).</li><li>Muramoto, T., Cannon, D., Gierlinski, M., Corrigan, A., Barton, G. J., Chubb, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+nascent+RNA+dynamics+reveals+distinct+types+of+transcriptional+pulse+regulation.">Live imaging of nascent RNA dynamics reveals distinct types of transcriptional pulse regulation.</a> Proc Natl Acad Sci USA. 109 (19), 7350-7355 (2012).</li><li>Ewers, H., Smith, A. E., Sbalzarini, I. F., Lilie, H., Koumoutsakos, P., Helenius, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-particle+tracking+of+murine+polyoma+virus-like+particles+on+live+cells+and+artificial+membranes.">Single-particle tracking of murine polyoma virus-like particles on live cells and artificial membranes.</a> Proc Natl Acad Sci USA. 102 (42), 15110-15115 (2005).</li><li>Forrest, K. M., Gavis, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+endogenous+RNA+reveals+a+diffusion+and+entrapment+mechanism+for+nanos+mRNA+localization+in+Drosophila.">Live imaging of endogenous RNA reveals a diffusion and entrapment mechanism for nanos mRNA localization in Drosophila.</a> Curr Biol. 13 (14), 1159-1168 (2003).</li><li>Weil, T. T., Forrest, K. M., Gavis, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+bicoid+mRNA+in+late+oocytes+is+maintained+by+continual+active+transport.">Localization of bicoid mRNA in late oocytes is maintained by continual active transport.</a> Dev Cell. 11 (2), 251-262 (2006).</li><li>Yamagishi, M., Ishihama, Y., Shirasaki, Y., Kurama, H., Funatsu, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+imaging+of+beta-actin+mRNAs+in+the+cytoplasm+of+a+living+cell.">Single-molecule imaging of beta-actin mRNAs in the cytoplasm of a living cell.</a> Exp Cell Res. 315 (7), 1142-1147 (2009).</li><li>Fusco, D., 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=Single+mRNA+molecules+demonstrate+probabilistic+movement+in+living+mammalian+cells.">Single mRNA molecules demonstrate probabilistic movement in living mammalian cells.</a> Curr Biol. 13 (2), 161-167 (2003).</li><li>Ozawa, T., Natori, Y., Sato, M., Umezawa, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+dynamics+of+endogenous+mitochondrial+RNA+in+single+living+cells.">Imaging dynamics of endogenous mitochondrial RNA in single living cells.</a> Nat Methods. 4 (5), 413-419 (2007).</li><li>Daigle, N., Ellenberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LambdaN-GFP:+an+RNA+reporter+system+for+live-cell+imaging.">LambdaN-GFP: an RNA reporter system for live-cell imaging.</a> Nat Methods. 4 (8), 633-636 (2007).</li><li>Bogaard, P. T., Tyagi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+molecular+beacons+to+study+dispersal+of+mRNPs+from+the+gene+locus.">Using molecular beacons to study dispersal of mRNPs from the gene locus.</a> Methods Mol Biol. 464, 91-103 (2009).</li><li>Vargas, D. Y., Raj, A., Marras, S. A., Kramer, F. R., Tyagi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+mRNA+transport+in+the+nucleus.">Mechanism of mRNA transport in the nucleus.</a> Proc Natl Acad Sci USA. 102 (47), 17008-17013 (2005).</li><li>Chen, A. K., Davydenko, O., Behlke, M. A., 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=Ratiometric+bimolecular+beacons+for+the+sensitive+detection+of+RNA+in+single+living+cells.">Ratiometric bimolecular beacons for the sensitive detection of RNA in single living cells.</a> Nucleic Acids Res. 38 (14), e148 (2010).</li><li>Zhang, X., 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=Quantitative+assessment+of+ratiometric+bimolecular+beacons+as+a+tool+for+imaging+single+engineered+RNA+transcripts+and+measuring+gene+expression+in+living+cells.">Quantitative assessment of ratiometric bimolecular beacons as a tool for imaging single engineered RNA transcripts and measuring gene expression in living cells.</a> Nucleic Acids Res. , (2013).</li><li>Tsourkas, A., Behlke, M. A., Bao, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure-function+relationships+of+shared-stem+and+conventional+molecular+beacons.">Structure-function relationships of shared-stem and conventional molecular beacons.</a> Nucleic Acids Res. 30 (19), 4208-4215 (2002).</li><li>Tsourkas, A., Behlke, M. A., Rose, S. D., Bao, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hybridization+kinetics+and+thermodynamics+of+molecular+beacons.">Hybridization kinetics and thermodynamics of molecular beacons.</a> Nucleic Acids Res. 31 (4), 1319-1330 (2003).</li><li>Bao, G., Rhee, W. J., 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=Fluorescent+probes+for+live-cell+RNA+detection.">Fluorescent probes for live-cell RNA detection.</a> Annu Rev Biomed Eng. 11, 25-47 (2009).</li><li>Zuker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mfold+web+server+for+nucleic+acid+folding+and+hybridization+prediction.">Mfold web server for nucleic acid folding and hybridization prediction.</a> Nucleic Acids Res. 31 (13), 3406-3415 (2003).</li><li>Chen, A. K., Behlke, M. A., 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=Efficient+cytosolic+delivery+of+molecular+beacon+conjugates+and+flow+cytometric+analysis+of+target+RNA.">Efficient cytosolic delivery of molecular beacon conjugates and flow cytometric analysis of target RNA.</a> Nucleic Acids Res. 36 (12), e69 (2008).</li><li>Zhang, X., 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=Quantitative+assessment+of+ratiometric+bimolecular+beacons+as+a+tool+for+imaging+single+engineered+RNA+transcripts+and+measuring+gene+expression+in+living+cells.">Quantitative assessment of ratiometric bimolecular beacons as a tool for imaging single engineered RNA transcripts and measuring gene expression in living cells.</a> Nucleic Acids Res. 41 (15), e152 (2013).</li><li>Ainger, 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=Transport+and+localization+of+exogenous+myelin+basic+protein+mRNA+microinjected+into+oligodendrocytes.">Transport and localization of exogenous myelin basic protein mRNA microinjected into oligodendrocytes.</a> J Cell Biol. 123 (2), 431-441 (1993).</li><li>Weil, T. T., Parton, R. M., Davis, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+the+message+clear:+visualizing+mRNA+localization.">Making the message clear: visualizing mRNA localization.</a> Trends Cell Biol. 20 (7), 380-390 (2010).</li><li>Paige, J. S., Wu, K. Y., Jaffrey, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+mimics+of+green+fluorescent+protein.">RNA mimics of green fluorescent protein.</a> Science. 333 (6042), 642-646 (2011).</li><li>Rackham, O., Brown, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+RNA-protein+interactions+in+living+cells:+FMRP+and+IMP1+interact+on+mRNAs.">Visualization of RNA-protein interactions in living cells: FMRP and IMP1 interact on mRNAs.</a> EMBO J. 23 (16), 3346-3355 (2004).</li><li>Valencia-Burton, M., McCullough, R. M., Cantor, C. R., Broude, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+visualization+in+live+bacterial+cells+using+fluorescent+protein+complementation.">RNA visualization in live bacterial cells using fluorescent protein complementation.</a> Nat Methods. 4 (5), 421-427 (2007).</li><li>Chen, A. K., Behlke, M. A., 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=Avoiding+false-positive+signals+with+nuclease-vulnerable+molecular+beacons+in+single+living+cells.">Avoiding false-positive signals with nuclease-vulnerable molecular beacons in single living cells.</a> Nucleic Acids Res. 35 (16), e105 (2007).</li><li>Chen, A. K., 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=Imaging+RNA+in+living+cells+with+molecular+beacons:+current+perspectives+and+challenges.">Imaging RNA in living cells with molecular beacons: current perspectives and challenges.</a> Journal of Innovative Optical Health Sciences. 2 (4), 315 (2009).</li><li>Nitin, N., Santangelo, P. J., Kim, G., Nie, S., Bao, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide-linked+molecular+beacons+for+efficient+delivery+and+rapid+mRNA+detection+in+living+cells.">Peptide-linked molecular beacons for efficient delivery and rapid mRNA detection in living cells.</a> Nucleic Acids Res. 32 (6), e58 (2004).</li><li>Santangelo, P. J., Nix, B., Tsourkas, A., Bao, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+FRET+molecular+beacons+for+mRNA+detection+in+living+cells.">Dual FRET molecular beacons for mRNA detection in living cells.</a> Nucleic Acids Res. 32 (6), e57 (2004).</li><li>Altman, R. B., 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=Cyanine+fluorophore+derivatives+with+enhanced+photostability.">Cyanine fluorophore derivatives with enhanced photostability.</a> Nat Methods. 9 (1), 68-71 (2012).</li><li>Kumaraswamy, 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=Fluorescent-conjugated+polymer+superquenching+facilitates+highly+sensitive+detection+of+proteases.">Fluorescent-conjugated polymer superquenching facilitates highly sensitive detection of proteases.</a> Proc Natl Acad Sci USA. 101 (20), 7511-7515 (2004).</li><li>Yang, C. J., Pinto, M., Schanze, K., Tan, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+synthesis+of+an+oligonucleotide-poly(phenylene+ethynylene)+conjugate+with+a+precise+one-to-one+molecular+ratio.">Direct synthesis of an oligonucleotide-poly(phenylene ethynylene) conjugate with a precise one-to-one molecular ratio.</a> Angew Chem Int Ed Engl. 44 (17), 2572-2576 (2005).</li><li>Lu, J., 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=Imaging+individual+microRNAs+in+single+mammalian+cells+in+situ.">Imaging individual microRNAs in single mammalian cells in situ.</a> Nucleic Acids Res. 37 (14), e100 (2009).</li><li>Raj, A., Peskin, C. S., Tranchina, D., Vargas, D. Y., Tyagi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stochastic+mRNA+synthesis+in+mammalian+cells.">Stochastic mRNA synthesis in mammalian cells.</a> PLoS Biol. 4 (10), e309 (2006).</li></ol>

使用传统的宽视场显微镜的能力，形象单一的工程RNA转录物在活细胞中，需要与每个RNA转录和低荧光背景与未结合的荧光探针发出的相关明亮，耐光荧光信号。在该方法中，明亮的荧光信号是由杂交多个（最多96个）基于寡核苷酸的荧光探针， 即 RBMBs，在每个RNA转录物来实现的。然而，仅仅四个绑定位点是足够16。集体信号产生，可以在实时的跟踪明亮的荧光斑点。多个探针的存在...

RNA转录物的表达调控是一个复杂的动态过程，主要是负责控制细胞的行为和命运。虽然RNA的口述细胞功能的重要性已经知道了一段时间，它往往是很难得出两个因为大多数RNA分析工具缺乏捕捉重要的调节事件，如转录爆破所需的空间和时间分辨率，RNA之间明确的连接贩卖人口和本地化RNA加工。这导致了一些技术，使个体的RNA转录物，以在实时1活细胞可视化的出现。也许，最突出的，这些技术利用了GFP-MS2融合蛋白靶向已被工程化以包含在3&#39;-UTR 2,3 MS2的结合位点的串联重复序列的RNA。通过将多个绿色荧光蛋白分子紧密接近，个别RNA转录出现明亮的荧光斑点通过荧光显微术。将GFP-MS2系统提供了前所未有的洞察RNA的行为，包括直接的可视化和转录的测量耐破4,5，检测独特的亚细胞定位和处理6-9，和RNA运输10的实时成像。然而，尽管GFP-MS2系统的巨大潜力，未结合的GFP-MS2融合蛋白可以创建限制了这种技术的通用性和动态范围的高背景荧光信号。几种方法已经被开发，以限制该背景信号，包括未结合的GFP-MS2 10的亚细胞区室化，蛋白片段互补11，并选择性RNA结合蛋白和目标12。然而，所有这些方法仍与RNA靶和GFP-MS2融合蛋白的相对和总表达敏感。 作为Alternative到GFP-MS2系统，分子信标也被用于检测工程化的RNA转录物的互补结合位点的3&#39;端非编码区13,14的串联重复序列。分子信标是被标记在一端与猝灭剂和在与荧光报道的另一端发夹形成的寡核苷酸探针。当未结合于靶R​​NA的荧光报道分子和猝灭剂保持在接近，从而导致低荧光状态。杂交时，该荧光报道分子和猝灭剂被强迫分开，荧光恢复。类似于GFP-MS2系统，多个分子信标结合到一个单一的RNA转录物的结果中，可以通过荧光显微镜来识别一个明亮的荧光斑点;然而，背景荧光预期要低的多，由于未结合的分子信标在骤冷结构。不幸的是，尽管巧妙活化机制并入米olecular信标设计方案，现在有越来越多的证据表明，未杂交的分子信标不留在发夹构象时引入活细胞。因此，它们产生假阳性信号，表明显著降低了信号 - 背景。为了克服这一缺点，我们最近开发了一种新的合成探针成像的RNA在活细胞中，比率双分子信标（RBMBs; 图1A）15,16。 RBMBs组成的构成的混合结构用从两个短发夹RNA（shRNA）和分子信标功能2 2&#39;-O-甲基寡核苷酸链。循环和荧光激活机制是类似的分子信标，而长的双链与3&#39;-UU突出域名的shRNA的多个特征。 shRNA的功能旨在推动核电出口，这是我们发现增加细胞内的生命周期，以&gt; ​​24小时，以最小的可观察到的退化，防止环路的非特异性开口。其结果RBMBs呈现显著更高的信号 - 背景比的分子信标。 但是应当注意的是，RBMBs不能使用的DNA寡核苷酸制备的，由于基于DNA的探针不具有相同的核输出能力，基于RNA的探针。在结构上，RBMB环通常被设计为15和21个碱基长之间，以创建经RNA杂交的特异性和选择性之间的平衡。短杆，其形成从两个自互补结构域通常被设计为4个碱基。如果较长的茎被选择，则RBMB环和靶RNA之间的杂交的速率显著减慢17,18。相反地​​，当选择了短茎序列的熔化温度通常太低，以维持茎 - 环结构，在37℃，从而导致高背景信号。该RBMB的特异性也红了uced作为干长度被缩短。由于RBMB茎-环也可以影响RBMB性能的序列，它必须仔细选择19。特别是，理想的循环序列应该具有最小的二级结构，杂交的RNA序列以最小的二级结构，避免蛋白质结合位点，并避免结合的脱靶。既RBMB和RNA二级结构的预测可以使用软件如MFOLD 20来获得。互补脱靶部位可使用核苷酸基本局部分配搜索工具（BLAST）确定。然而，因为在模型预测和确定蛋白质伺机网站难度不一致的，所有RBMBs的特异性，最终必须通过实验验证。 如果需要的话，一个非淬火参考染料是不敏感的杂交状态可以被添加到RBMB 15。又多了一个参考染料可公关ovide的标记探针递送和用于比例成像，如果细胞总荧光的更精确的测量是必需的。参考染料允许测量要调节为由于细胞 - 细胞间的变化在传递中背景的差异。然而，成像个体的RNA时誊基准染料是没有必要的。值得注意的是，一些参考染料可以与RBMBs的离核的出口干扰，导致在细胞核中稍高的背景信号。 当设计得当，RBMBs可用于图像个体的RNA转录物在单个活细胞中，如果靶RNA被工程化以包含至少四个RBMB结合位点（ 图1B）16。 RBMBs能够高效率地递送到大范围通过微穿孔的细胞类型的，很少或几乎没有作用，对细胞活力21，和基因表达的定量测量可在30分钟内获得的。此外，该方法是相当不敏感RBMB浓度和靶RNA水平，由于来自未结合RBMBs荧光被有效地淬灭。这里我们提供了用于制备和纯化RBMBs的方法的详细描述，以及用于递送RBMBs进入活细胞通过微穿孔的一般过程和单RNA转录物中的实时成像。

<table border="0" cellpadding="0" cellspacing="0" width="977">
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real-time imaging of single engineered rna transcripts in living cells using ratiometric bimolecular beacons

人们越来越认识到这两个基因表达的时空调控可能对细胞功能的重要后果，导致了不同的技术的发展，以可视个体的RNA转录物在单个活细胞。最近已经描述的一个有前途的技术利用的寡核苷酸为基础的光学探头，比率双分子信标（RBMB），以检测该被工程化以包含在3&#39;-端非翻译区的RBMB靶序列的至少四个串联重复序列的RNA转录物。 RBMBs是专门设计成发射后，杂交到互补RNA明亮的荧光信号，但在其他方面仍骤冷。在这种方法中，使用合成探针允许耐光的，红移，并且将用于成像高度发光有机染料。多RBMBs的宽视场荧光显微镜下观察时，结合改造的RNA转录产生离散的荧光斑点。因此，个体的RNA转录物的运动可以容易地通过取一个时间序列的荧光图像的可视化中的实时性。在这里，我们描述RBMBs的制备和纯化，递送到细胞微穿孔和活细胞的单个RNA转录物成像。

不久下列HT1080细胞中RBMBs的存在下的微穿孔，被改造的那个人的RNA转录物，以含有多个RBMB结合位点在其3&#39;-UTR时通过宽视场荧光显微镜（ 图2）拍摄显示为明亮的荧光斑点。而个体的RNA转录物与少至4 RBMB结合位点可以在活细胞进行成像，越RBMB结合在3&#39;-UTR部位，越强的荧光信号。而且，越是RBMBs绑定到每个成绩单的时间越长的RNA可以可视化前的信号丢失，由于漂白。在这个协议中，R...

Watch this Scientific Journal Video about Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons at JoVE.com

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

单工程的RNA转录物的活细胞实时成像采用比例式双分子信标

比例式双分子信标（RBMBs）可用于将图像的单个设计RNA转录活细胞中。在这里，我们描述RBMBs，交货RBMBs到细胞通过微穿孔和的单个RNA转录物中的实时荧光成像的制备和纯化。

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Research

JoVE Journal

Biology

Imaging Cell Shape Change in Living Drosophila Embryos

Imaging Cell Shape Change in Living Drosophila Embryos

影像生活细胞的形状变化果蝇胚胎

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

科研

生物学

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

确定基因表达概况 C。线虫使用微阵列和实时PCR

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, ...

Measuring the Kinetics of mRNA Transcription in Single Living Cells

测量单个活细胞中的mRNA转录的动力学

The transcriptional activity of RNA polymerase II (Pol II) is a dynamic process and therefore measuring the kinetics of the transcriptional process in ...

FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

FRET显微镜在活细胞单分子生物传感器的信号事件的实时监测

F&ouml;rster resonance energy transfer (FRET) microscopy  continues to gain increasing interest as a technique for real-time monitoring  of biochemical ...

Quantitative, Real-time Analysis of Base Excision Repair Activity in Cell Lysates Utilizing Lesion-specific Molecular Beacons

定量，实时分析，利用分子信标病​​变特定的细胞裂解液中的碱基切除修复活动

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates using base ...

Fluorescence Imaging with One-nanometer Accuracy (FIONA)

Fluorescence Imaging with One-nanometer Accuracy FIONA

荧光成像与一纳米精度（FIONA）

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

高分辨率成像定时，并在活体人脐静脉内皮细胞微管动力学自动分析

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

Application of Genetically Encoded Fluorescent Nitric Oxide (NO&#8226;) Probes, the geNOps, for Real-time Imaging of NO&#8226; Signals in Single Cells

Application of Genetically Encoded Fluorescent Nitric Oxide NOÃƒÆ’Ã‚Â¢ÃƒÂ¢Ã¢â‚¬Å¡Ã‚Â¬Ãƒâ€šÃ‚Â¢ Probes, the geNOps, for Real-time Imaging of NOÃƒÆ’Ã‚Â¢ÃƒÂ¢Ã¢â‚¬Å¡Ã‚Â¬Ãƒâ€šÃ‚Â¢ Signals in Single Cells

遗传编码的荧光一氧化氮的应用（NO•）探针，所述geNOps，用于在单细胞NO•信号的实时影像

Nitric Oxide (NO&#8226;) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence ...

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

单分子荧光原位杂交实验中单个大肠埃希菌细胞的转录标记方法

A method is described for labeling individual messenger RNA (mRNA) transcripts in fixed bacteria for use in single-molecule fluorescence in situ ...

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

利用两色单分子荧光在原位杂交中Escherichia coli探索大肠杆菌中空间和时间中的 mRNA 动力学

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an ...