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

Dette arbejde blev støttet af National Science Foundation KARRIERE Award (0953583) og National Institute of Health NCI / R21-CA116102, NCI / R21-CA125088, NIBIB / R01-EB012065, NCI / R01-CA157766.

I denne protokol blev en oligonucleotid (RBMB1) mærket på dets 5&#39;-enden med en CF640R reporter farvestof og har sekvensen: 5&#39;mCmUmUmC mGmUmC mCmAmC mAmAmA mCmAmC mAmAmC mUmCmC mU mGmAmAmG mGmAmC mGmGmC mAmGmC mGmUmG mCmAmG mCmUmC MUMU-3 &#39;. Selvstændige komplementære domæner, der driver dannelsen af ​​hårnålen struktur, er med fed skrift. Det andet oligonukleotid (RBMB2) blev mærket ved 3&#39;-enden med en Iowa Sort RQ-Sp quencheren og har sekvensen: 5&#39;-mGmAmG...

<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>

Evnen til at afbilde enkelt manipuleret RNA-transkripter i levende celler ved hjælp af en konventionel lang-felt mikroskop kræver en lys og fotostabile fluorescerende signal at være forbundet med hver RNA transcript og en lav fluorescerende baggrund stammer fra ubundne fluorescerende prober. Ved denne fremgangsmåde er en lys fluorescerende signal opnået ved hybridisering flere (op til 96) oligonukleotid-baserede fluorescerende prober, dvs RBMBs, på hver RNA-transkript. Men så få som fire bindinger stede...

Udtrykket og regulering af RNA-transkripter er en kompleks og dynamisk proces, der er i høj grad ansvarlig for at kontrollere celle adfærd og skæbne. Selvom betydningen af ​​RNA i diktere cellefunktion har været kendt i nogen tid, er det ofte vanskeligt at drage klare forbindelser mellem de to, da de fleste RNA-analyseværktøjer mangler den rumlige og tidsmæssige opløsning kræves for at optage vigtige regulerende begivenheder såsom transskription brister, RNA menneskehandel, og lokaliserede RNA-behandling. Dette har ført til fremkomsten af flere teknikker, der tillader individuelle RNA-transkripter, der skal visualiseres i levende celler i realtid 1. Måske den mest fremtrædende af disse teknikker anvender et GFP-MS2-fusionsproteinet til at målrette RNA, der er blevet manipuleret til at indeholde tandemgentagelser af MS2-bindingssted i 3&#39;-UTR 2,3. Ved at bringe flere GFP-molekyler i tæt nærhed, individuelle RNA-transkripter fremstå som lyse fluorescerende plettervia fluorescensmikroskopi. GFP-MS2-system har givet en hidtil uset indsigt i RNA adfærd, herunder direkte visualisering og målinger af transkriptionelle sprængfyldt 4,5, påvisning af unikke sub-cellulære lokalisering og behandling 6-9, og real-time scanning af RNA-transport 10. Trods det enorme potentiale af GFP-MS2 system kan ubundne GFP-MS2-fusionsproteiner skabe en høj baggrund fluorescerende signal, der begrænser alsidigheden og dynamikområde af denne teknik. Adskillige fremgangsmåder er blevet udviklet til at begrænse denne baggrund signal, herunder subcellulære opdeling af ubundet GFP-MS2 10 proteinfragment komplementation 11, og alternative RNA-bindende proteiner og mål 12. Men alle disse fremgangsmåder forbliver følsomme over for den relative og totale ekspression af RNA-mål og GFP-MS2 fusionsprotein. Som etlternative til GFP-MS2-systemer, har molekylære beacons også blevet anvendt til at påvise manipuleret RNA-transkripter med tandemgentagelser af den komplementære bindingssted i 3&#39;-UTR 13,14. Molecular beacons er hårnåle-dannende oligonukleotidprober, der er mærket i den ene ende med en quencher og ved den anden ende med et fluorescerende reporter. Når den ikke er forpligtet til at målrette RNA den fluorescerende reporter og quencher forblive i umiddelbar nærhed, hvilket resulterer i en lav-fluorescerende tilstand. Efter hybridisering den fluorescerende reporter og quencheren tvunget fra hinanden og fluorescens er genoprettet. Svarende til GFP-MS2-system, binding af flere molekylære beacons på en enkelt RNA-transkript resulterer i en stærkt fluorescerende plet, der kan identificeres ved hjælp af fluorescens mikroskopi; imidlertid er baggrundsfluorescens forventes at være meget lavere på grund af den bratkølede konfigurationen af ​​ubundne molekylære beacons. Til trods den smarte aktiveringsmekanisme, der er indeholdt i deolecular beacon design, er der nu voksende beviser for, at ikke-hybridiseret molekylære beacons ikke forbliver i hårnål kropsbygning når det indføres i levende celler. Derfor er de genererer et falsk positivt signal, der reducerer signal-til-baggrund. For at overvinde denne mangel, vi for nylig udviklet et nyt syntetisk sonde til billeddannelse RNA i levende celler Ratiometrisk bimolekylære beacons (RBMBs; figur 1a) 15,16. RBMBs er sammensat af to 2&#39;-O-methyl-oligonukleotid strenge, der danner en hybrid struktur med egenskaber fra både korte hårnål RNA (shRNA) og molekylære beacons. Sløjfen og fluorescensaktivering mekanisme svarer til den molekylære beacon, mens den lange dobbeltstrenget domæne med en 3&#39;-UU overhæng er mere karakteristisk for shRNA. De shRNA funktioner er designet til at køre nuklear eksport, som vi har fundet stigninger intracellulær levetid til&gt; 24 timer, med minimal observerbare nedbrydning, oghindrer uspecifik åbning af sløjfen. Som et resultat RBMBs udviser en signifikant højere signal-baggrund end molekylære beacons. Det skal bemærkes, at RBMBs ikke kan fremstilles ved hjælp af DNA-oligonukleotider, da DNA-baserede prober ikke besidder de samme kernevåbenkapacitet eksport som RNA-baserede prober. Strukturelt er RBMB løkke typisk designet til at være mellem 15 og 21 baser lange, for at skabe en balance mellem specificitet og selektivitet ved RNA hybridisering. Den korte stilk, der udgør de to selvstændige komplementære domæner er normalt designet til at være 4 baser. Hvis der vælges en længere stilk, er hastigheden af hybridisering mellem RBMB loop og mål-RNA betydeligt langsommere 17,18. Omvendt, når en kortere stamceller sekvens er valgt smeltetemperaturen ofte er for lav til at opretholde stilk-loop-struktur ved 37 ° C, hvilket fører til en høj baggrund signal. Specificiteten af ​​RBMB er også røduced som stilken længde forkortes. Da sekvensen af RBMB stilk-sløjfe kan også påvirke RBMB præstationer, skal udvælges omhyggeligt 19. Især bør ideelle loop sekvenser har minimal sekundær struktur, hybridisere til RNA-sekvenser med minimal sekundær struktur, undgå proteinbindingssteder og undgå off-target-bindende. Forudsigelser af både RBMB og RNA sekundær struktur kan opnås ved hjælp af software som f.eks mfold 20. Supplerende off-target sites kan identificeres ved hjælp af et nukleotid Basic Local Assignment søgeværktøj (BLAST). Men på grund af uoverensstemmelser i modellens forudsigelser og det er vanskeligt at identificere protein-biding websteder, specificitet alle RBMBs sidste ende skal valideres eksperimentelt. Om ønsket kan en uquenchet henvisning farvestof, der er ufølsom over for hybridisering tilstand sættes til RBMB 15. Tilføjelsen af ​​en reference farvestof kan PRovide en markør for sonde levering og bruges til ratiometrisk billeddannelse, hvis der kræves mere nøjagtige målinger af totale cellulære fluorescens. Referenceværdierne farvestoffer tillader målinger, der skal justeres for forskelle i baggrunden på grund af celle-til-celle variation i leveringen. Men når billeddannelse individuelle RNA-transkripter, reference farvestof er ikke nødvendig. Især kan nogle reference farvestoffer interferere med eksport af RBMBs fra kernen, hvilket fører til en lidt højere baggrund signal i kernen. Når konstrueret korrekt, kan RBMBs anvendes til at afbilde individuelle RNA-transkripter i enkelte levende celler, hvis målet RNA manipuleret til at indeholde mindst fire RBMB bindingssteder (figur 1B) 16. RBMBs effektivt kan leveres i en lang række af celletyper via mikroperforering, med lidt eller ingen virkning på cellelevedygtighed 21, og kvantitative målinger af genekspression kan væreerhvervet inden for 30 min. Desuden metode er forholdsvis ufølsom over for RBMB koncentration og mål-RNA-niveauer, eftersom fluorescens fra ubundne RBMBs effektivt er standset. Her giver vi en detaljeret beskrivelse af den anvendte metode til at forberede og rense RBMBs, samt en generel procedure for levering af RBMBs ind i levende celler via mikroporering og billeddannelse af enkelt RNA-transkripter i real-tid.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Nuclease-free Water</td>
			<td style="width:207px;">Life Technologies</td>
			<td style="width:220px;">AM9932</td>
			<td style="width:244px;">no DEPC-treated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">DPBS</td>
			<td style="width:207px;">Life Technologies</td>
			<td>14190-144</td>
			<td>No Ca2+ and Mg2+</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Cary 100 UV-Vis Spectrophotometer</td>
			<td style="width:207px;">Agilent Technologies</td>
			<td style="width:220px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Potassium Phosphate Dibasic</td>
			<td style="width:207px;">Fisher Scientific</td>
			<td style="width:220px;">P290-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Potassium Phosphate Monobasic</td>
			<td style="width:207px;">Fisher Scientific</td>
			<td style="width:220px;">P284-500&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Sodium Phosphate Monobasic</td>
			<td style="width:207px;">Fisher Scientific</td>
			<td style="width:220px;">S381-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Microcentrifuge tubes</td>
			<td style="width:207px;">Eppendorf</td>
			<td style="width:220px;">22364111</td>
			<td>1.5mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Chromatography Column</td>
			<td style="width:207px;">Kimble Chase Life Science</td>
			<td style="width:220px;">420400-0720</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Superdex 75 Prep Grade</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Syringe Pump</td>
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			<td style="width:220px;">BS-300</td>
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		</tr>
		<tr height="21">
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			<td style="width:220px;">301035</td>
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		<tr height="21">
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			<td style="width:220px;">UFC501096</td>
			<td>Mw 10,000 cutoff</td>
		</tr>
		<tr height="21">
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		</tr>
		<tr height="21">
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			<td style="width:220px;">P7280-5MG</td>
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		<tr height="21">
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			<td style="width:220px;">155409</td>
			<td>Working volume 0.2-0.5mL</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:307px;">HT1080</td>
			<td style="width:207px;">ATCC</td>
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		<tr height="21">
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			<td>25cm2</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:307px;">DMEM</td>
			<td style="width:207px;">Life Technologies</td>
			<td style="width:220px;">11965084</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">DMEM without Phenol Red</td>
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			<td style="width:220px;">21063029</td>
			<td>High glucose</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Fetal Bovine Serum</td>
			<td style="width:207px;">Sigma-Aldrich</td>
			<td style="width:220px;">F2442-500ML&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Penecillin/Streptomycin</td>
			<td style="width:207px;">Life Technologies</td>
			<td style="width:220px;">15140122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Trypsin</td>
			<td style="width:207px;">Life Technologies</td>
			<td style="width:220px;">25300054</td>
			<td>0.05% Trypsin-EDTA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Neon transfection system</td>
			<td style="width:207px;">Life Technologies</td>
			<td style="width:220px;">MPK5000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Neon transfection system kit&#160;</td>
			<td style="width:207px;">Life Technologies</td>
			<td style="width:220px;">MPK1096</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Hemacytometer</td>
			<td style="width:207px;">Fisher Scientific</td>
			<td style="width:220px;">02-671-10&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoescht 33342</td>
			<td style="width:207px;">Life Technologies</td>
			<td style="width:220px;">H1399</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">IX-81 Inverted fluorescence microscope&#160;</td>
			<td style="width:207px;">Olympus</td>
			<td style="width:220px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">SOLA light engine</td>
			<td style="width:207px;">Lumencor</td>
			<td style="width:220px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Metamorph</td>
			<td style="width:207px;">Molecular Devices</td>
			<td style="width:220px;"></td>
			<td>Software controlling microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immersol oil 518F</td>
			<td style="width:207px;">Fisher Scientific</td>
			<td style="width:220px;">12-624-66B&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

real-time imaging of single engineered rna transcripts in living cells using ratiometric bimolecular beacons

Den voksende erkendelse af, at både den tidsmæssige og rumlige regulering af genekspression kan have betydelige konsekvenser for cellefunktion har ført til udviklingen af ​​forskellige teknikker til at visualisere de enkelte RNA-transkripter i enkelte levende celler. En lovende teknik, der for nylig blevet beskrevet benytter en oligonucleotid-baseret optisk sonde, ratiometrisk bimolekylær beacon (RBMB), til påvisning af RNA-transkripter, der var manipuleret til at indeholde mindst fire tandemgentagelser af RBMB målsekvensen i 3&#39;-utranslaterede region. RBMBs er specielt konstrueret til at udsende en lys fluorescerende signal ved hybridisering til komplementært RNA, men ellers forbliver standset. Anvendelsen af ​​en syntetisk probe i denne fremgangsmåde giver fotostabile, rød-skiftede og stærkt emitterende organiske farvestoffer, der skal anvendes til billeddannelse. Binding af flere RBMBs til de konstruerede RNA-transkripter resulterer i diskrete fluorescenspletter når de ses under et bredt felt fluorescerende mikroskop. Derfor kan bevægelsen af ​​individuelle RNA-transkripter let visualiseres i realtid ved at tage en serie af fluorescerende billeder tid. Her beskriver vi fremstilling og oprensning af RBMBs, levering i celler ved mikroporering og leve-cell imaging af enkelte RNA-transkripter.

Kort efter mikroperforeringsanordningen af HT1080-celler i nærværelse af RBMBs, individuelle RNA-transkripter, der blev manipuleret indeholde flere RBMB bindingssites i deres 3&#39;-UTR synes som lyse fluorescerende pletter, når afbildet med en bredt område fluorescensmikroskopi (Figur 2). Mens individuelle RNA-transkripter med så få som fire RBMB bindingssteder kan afbildes i levende-celler, mere RBMB bindingssteder i 3&#39;-UTR, jo stærkere det fluorescerende signal. Desuden, jo flere RBMBs der...

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

Real-time Imaging af Single Engineered RNA-transkripter i levende celler Brug Ratiometrisk bimolekylær Beacons

Ratiometrisk bimolekylære beacons (RBMBs) kan anvendes til at afbilde enkelt manipuleret RNA-transkripter i levende celler. Her beskriver vi forberedelse og rensning af RBMBs, levering af RBMBs i celler ved mikroporering og fluorescerende billeddannelse af enkelt RNA-transkripter i real-tid.

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

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

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

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

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 mikroskopi af Real-time overvågning af signalering begivenheder i levende celler Brug unimolekylær Biosensorer

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

Kvantitativ, Real-time analyse af base excision reparation aktivitet i cellelysater Udnyttelse Læsionssynligheden-specifikke Molecular Beacons

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

Fluorescensimagografi med One-nanometer Nøjagtighed (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

Høj opløsning Time-lapse Imaging og Automatiseret analyse af mikrotubulusdynamik i levende menneske navlestrengsveneendotelceller

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

Anvendelse af genetisk indkodede fluorescerende nitrogenoxid (NO •) Probes, de geNOps, for Real-time Imaging af NO • Signaler i enkeltceller

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

Metode for mærkning udskrifter i individuelle Escherichia coli celler for enkelt-molekyle fluorescens In Situ hybridisering eksperimenter

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

Sondering mRNA kinetik i rum og tid i Escherichia coli ved hjælp af to-color single-molecule fluorescens In Situ Hybridization

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