Name: use of alu element containing minigenes to analyze circular rnas
Uploaded: 2020-03-10T12:00:00
Description: Watch this Scientific Journal Video about Use of Alu Element Containing Minigenes to Analyze Circular RNAs at JoVE.com

Bu çalışma Savunma Bakanlığı DoD hibe AZ180075 tarafından desteklenmiştir. Stefan Stamm teşekkürler Jacqueline Noonan Vakfı. Anna Pawluchin DAAD, Alman akademik değişim programı tarafından desteklenen, Justin R. Welden University of Kentucky Max Steckler Ödülü'nü aldı.

1. Yapıların tasarımı
<ol><li>Dairesel RNA oluşumu için gerekli tekrarlayan elementleri belirlemek ve bunları yapılara dahil etmek için UCSC genomtarayıcısı 24'ü kullanın. Daha da önemlisi, amplifikasyon için astartekrarlayan elemanların dışında olması gerekir.</li>
	<li>Dairesel RNA dizisini(Tamamlayıcı Şekil 1 bir test dizisidir) <a href="https://genome.ucsc.edu/cgi-bin/hgBlat?command=start" target="_blank">https://genome.ucsc.edu/cgi-bin/hgBlat?command...

<ol>
	<li>Jeck, W. R., 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=Circular+RNAs+are+abundant,+conserved,+and+associated+with+ALU+repeats.">Circular RNAs are abundant, conserved, and associated with ALU repeats.</a> RNA. 19 (2), 141-157 (2013).</li><li>Zhang, X. O., 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=Complementary+sequence-mediated+exon+circularization.">Complementary sequence-mediated exon circularization.</a> Cell. 159 (1), 134-147 (2014).</li><li>Deininger, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alu+elements:+know+the+SINEs.">Alu elements: know the SINEs.</a> Genome Biology. 12 (12), 236 (2011).</li><li>Bazak, L., Levanon, E. Y., Eisenberg, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+of+Alu+editability.">Genome-wide analysis of Alu editability.</a> Nucleic Acids Research. 42 (11), 6876-6884 (2014).</li><li>Levanon, E. Y., 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=Systematic+identification+of+abundant+A-to-I+editing+sites+in+the+human+transcriptome.">Systematic identification of abundant A-to-I editing sites in the human transcriptome.</a> Nature Biotechnology. 22 (8), 1001-1005 (2004).</li><li>Hansen, T. 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=Natural+RNA+circles+function+as+efficient+microRNA+sponges.">Natural RNA circles function as efficient microRNA sponges.</a> Nature. 495 (7441), 384-388 (2013).</li><li>Kelly, S., Greenman, C., Cook, P. R., Papantonis, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exon+Skipping+Is+Correlated+with+Exon+Circularization.">Exon Skipping Is Correlated with Exon Circularization.</a> Journal of Molecular Biology. 427 (15), 2414-2417 (2015).</li><li>Li, Z., 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=Exon-intron+circular+RNAs+regulate+transcription+in+the+nucleus.">Exon-intron circular RNAs regulate transcription in the nucleus.</a> Nature Structural &amp; Molecular Biology. 22 (3), 256-264 (2015).</li><li>Yang, Y., 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=Extensive+translation+of+circular+RNAs+driven+by+N(6)-methyladenosine.">Extensive translation of circular RNAs driven by N(6)-methyladenosine.</a> Cell Research. 27 (6), 626-641 (2017).</li><li>Abe, 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=Rolling+Circle+Translation+of+Circular+RNA+in+Living+Human+Cells.">Rolling Circle Translation of Circular RNA in Living Human Cells.</a> Scientific Reports. 5, 16435 (2015).</li><li>Salzman, J., Chen, R. E., Olsen, M. N., Wang, P. L., Brown, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-type+specific+features+of+circular+RNA+expression.">Cell-type specific features of circular RNA expression.</a> PLOS Genetics. 9 (9), 1003777 (2013).</li><li>Ottesen, E. W., Luo, D., Seo, J., Singh, N. N., Singh, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Survival+Motor+Neuron+genes+generate+a+vast+repertoire+of+circular+RNAs.">Human Survival Motor Neuron genes generate a vast repertoire of circular RNAs.</a> Nucleic Acids Research. 47 (6), 2884-2905 (2019).</li><li>Stoss, O., Stoilov, P., Hartmann, A. M., Nayler, O., Stamm, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+in+vivo+minigene+approach+to+analyze+tissue-specific+splicing.">The in vivo minigene approach to analyze tissue-specific splicing.</a> Brain Research Protocols. 4, 383-394 (1999).</li><li>Mardon, H. J., Sebastio, G., Baralle, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+exon+sequences+in+alternative+splicing+of+the+human+fibronectin+gene.">A role for exon sequences in alternative splicing of the human fibronectin gene.</a> Nucleic Acids Research. 15, 7725-7733 (1987).</li><li>Gaildrat, P., 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=Use+of+splicing+reporter+minigene+assay+to+evaluate+the+effect+on+splicing+of+unclassified+genetic+variants.">Use of splicing reporter minigene assay to evaluate the effect on splicing of unclassified genetic variants.</a> Methods in Molecular Biology. 653, 249-257 (2010).</li><li>Cooper, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+minigene+systems+to+dissect+alternative+splicing+elements.">Use of minigene systems to dissect alternative splicing elements.</a> Methods. 37 (4), 331-340 (2005).</li><li>Baralle, D., Baralle, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Splicing+in+action:+assessing+disease+causing+sequence+changes.">Splicing in action: assessing disease causing sequence changes.</a> Journal of Medical Genetics. 42 (10), 737-748 (2005).</li><li>Percifield, R., Murphy, D., Stoilov, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medium+throughput+analysis+of+alternative+splicing+by+fluorescently+labeled+RT-PCR.">Medium throughput analysis of alternative splicing by fluorescently labeled RT-PCR.</a> Methods in Molecular Biology. 1126, 299-313 (2014).</li><li>Stoilov, P., Lin, C. H., Damoiseaux, R., Nikolic, J., Black, D. 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+high-throughput+screening+strategy+identifies+cardiotonic+steroids+as+alternative+splicing+modulators.">A high-throughput screening strategy identifies cardiotonic steroids as alternative splicing modulators.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (32), 11218-11223 (2008).</li><li>Shen, M., 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=Pyrvinium+pamoate+changes+alternative+splicing+of+the+serotonin+receptor+2C+by+influencing+its+RNA+structure.">Pyrvinium pamoate changes alternative splicing of the serotonin receptor 2C by influencing its RNA structure.</a> Nucleic Acids Research. 41 (6), 3819-3832 (2013).</li><li>Noto, J. J., Schmidt, C. A., Matera, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+and+expressing+circular+RNAs+via+tRNA+splicing.">Engineering and expressing circular RNAs via tRNA splicing.</a> RNA Biology. , 1-7 (2017).</li><li>Schmidt, C. A., Noto, J. J., Filonov, G. S., Matera, A. 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+Method+for+Expressing+and+Imaging+Abundant,+Stable,+Circular+RNAs+In+Vivo+Using+tRNA+Splicing.">A Method for Expressing and Imaging Abundant, Stable, Circular RNAs In Vivo Using tRNA Splicing.</a> Methods in Enzymology. 572, 215-236 (2016).</li><li>Welden, J. R., van Doorn, J., Nelson, P. T., Stamm, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+MAPT+locus+generates+circular+RNAs.">The human MAPT locus generates circular RNAs.</a> Biochimica et Biophysica Acta - Molecular Basis of Disease. , 2753-2760 (2018).</li><li>Casper, 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=The+UCSC+Genome+Browser+database:+2018+update.">The UCSC Genome Browser database: 2018 update.</a> Nucleic Acids Research. 46, 762-769 (2018).</li><li>Grozdanov, P. N., MacDonald, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+plasmid+vectors+expressing+FLAG-tagged+proteins+under+the+regulation+of+human+elongation+factor-1alpha+promoter+using+Gibson+assembly.">Generation of plasmid vectors expressing FLAG-tagged proteins under the regulation of human elongation factor-1alpha promoter using Gibson assembly.</a> Journal of Visualized Experiments. (96), (2015).</li><li>Wang, Y., 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+novel+strategy+to+engineer+DNA+polymerases+for+enhanced+processivity+and+improved+performance+in+vitro.">A novel strategy to engineer DNA polymerases for enhanced processivity and improved performance in vitro.</a> Nucleic Acids Research. 32 (3), 1197-1207 (2004).</li><li>Gibson, D. G., 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=Enzymatic+assembly+of+DNA+molecules+up+to+several+hundred+kilobases.">Enzymatic assembly of DNA molecules up to several hundred kilobases.</a> Nature Methods. 6 (5), 343-345 (2009).</li><li>Conn, S. 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=The+RNA+binding+protein+quaking+regulates+formation+of+circRNAs.">The RNA binding protein quaking regulates formation of circRNAs.</a> Cell. 160 (6), 1125-1134 (2015).</li><li>Jeck, W. R., Sharpless, 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=Detecting+and+characterizing+circular+RNAs.">Detecting and characterizing circular RNAs.</a> Nature Biotechnology. 32 (5), 453-461 (2014).</li><li>Pamudurti, N. R., 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=Translation+of+CircRNAs.">Translation of CircRNAs.</a> Molecular Cell. 66 (1), 9-21 (2017).</li><li>Kramer, M. C., 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=Combinatorial+control+of+Drosophila+circular+RNA+expression+by+intronic+repeats,+hnRNPs,+and+SR+proteins.">Combinatorial control of Drosophila circular RNA expression by intronic repeats, hnRNPs, and SR proteins.</a> Genes &amp; Development. 29 (20), 2168-2182 (2015).</li><li>Starke, 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=Exon+circularization+requires+canonical+splice+signals.">Exon circularization requires canonical splice signals.</a> Cell Reports. 10 (1), 103-111 (2015).</li><li>Suzuki, H., Tsukahara, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+view+of+pre-mRNA+splicing+from+RNase+R+resistant+RNAs.">A view of pre-mRNA splicing from RNase R resistant RNAs.</a> International Journal of Molecular Sciences. 15 (6), 9331-9342 (2014).</li><li>Zhang, X. O., 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=Diverse+alternative+back-splicing+and+alternative+splicing+landscape+of+circular+RNAs.">Diverse alternative back-splicing and alternative splicing landscape of circular RNAs.</a> Genome Research. 26 (9), 1277-1287 (2016).</li><li>Liang, D., Wilusz, 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=Short+intronic+repeat+sequences+facilitate+circular+RNA+production.">Short intronic repeat sequences facilitate circular RNA production.</a> Genes &amp; Development. 28 (20), 2233-2247 (2014).</li><li>Wang, Y., Mandelkow, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tau+in+physiology+and+pathology.">Tau in physiology and pathology.</a> Nature Reviews Neuroscience. 17 (1), 5-21 (2016).</li><li>Fejes-Toth, 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=Post-transcriptional+processing+generates+a+diversity+of+5'-modified+long+and+short+RNAs.">Post-transcriptional processing generates a diversity of 5'-modified long and short RNAs.</a> Nature. 457 (7232), 1028-1032 (2009).</li><li>Gao, Q. 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=Complex+regulation+of+tau+exon+10,+whose+missplicing+causes+frontotemporal+dementia.">Complex regulation of tau exon 10, whose missplicing causes frontotemporal dementia.</a> Journal of Neurochemistry. 74 (2), 490-500 (2000).</li><li>Hartmann, A. M., 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=Regulation+of+alternative+splicing+of+human+tau+exon+10+by+phosphorylation+of+splicing+factors.">Regulation of alternative splicing of human tau exon 10 by phosphorylation of splicing factors.</a> Molecular and Cellular Neuroscience. 18 (1), 80-90 (2001).</li><li>Wang, Y., Wang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+backsplicing+produces+translatable+circular+mRNAs.">Efficient backsplicing produces translatable circular mRNAs.</a> RNA. 21 (2), 172-179 (2015).</li><li>Yang, Y., Wang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Constructing+GFP-Based+Reporter+to+Study+Back+Splicing+and+Translation+of+Circular+RNA.">Constructing GFP-Based Reporter to Study Back Splicing and Translation of Circular RNA.</a> Methods in Molecular Biology. 1724, 107-118 (2018).</li><li>Zheng, Q., 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=Circular+RNA+profiling+reveals+an+abundant+circHIPK3+that+regulates+cell+growth+by+sponging+multiple+miRNAs.">Circular RNA profiling reveals an abundant circHIPK3 that regulates cell growth by sponging multiple miRNAs.</a> Nature Communications. 7, 11215 (2016).</li><li>Jia, W., Xu, B., Wu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circular+RNA+expression+profiles+of+mouse+ovaries+during+postnatal+development+and+the+function+of+circular+RNA+epidermal+growth+factor+receptor+in+granulosa+cells.">Circular RNA expression profiles of mouse ovaries during postnatal development and the function of circular RNA epidermal growth factor receptor in granulosa cells.</a> Metabolism. 85, 192-204 (2018).</li><li>Liang, 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=The+Output+of+Protein-Coding+Genes+Shifts+to+Circular+RNAs+When+the+Pre-mRNA+Processing+Machinery+Is+Limiting.">The Output of Protein-Coding Genes Shifts to Circular RNAs When the Pre-mRNA Processing Machinery Is Limiting.</a> Molecular Cell. 68 (5), 940-954 (2017).</li><li>Legnini, I., 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=Circ-ZNF609+Is+a+Circular+RNA+that+Can+Be+Translated+and+Functions+in+Myogenesis.">Circ-ZNF609 Is a Circular RNA that Can Be Translated and Functions in Myogenesis.</a> Molecular Cell. 66 (1), 22-37 (2017).</li><li>Li, 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=Coordinated+circRNA+Biogenesis+and+Function+with+NF90/NF110+in+Viral+Infection.">Coordinated circRNA Biogenesis and Function with NF90/NF110 in Viral Infection.</a> Molecular Cell. 67 (2), 214-227 (2017).</li><li>Zhang, Y., 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=The+Biogenesis+of+Nascent+Circular+RNAs.">The Biogenesis of Nascent Circular RNAs.</a> Cell Reports. 15 (3), 611-624 (2016).</li></ol>

Genel olarak, dairesel RNA'lar düşükboldur 1, bu da onların fonksiyon ve oluşumunun incelenmesini zorlaştırır. Lineer RNA13benzer , muhabir minigenlerin kullanımı dairesel RNA oluşumunu düzenleyen cis ve trans-etkili faktörlerin belirlenmesini sağlar. Böylece, bu yaklaşım daha da endojen genler kullanılarak test edilebilir hipotezler oluşturur.
En kritik adım muhabir genin tasarımıdır. DNA parçalarının enzimatik montajı...

Dairesel RNA'lar Dairesel RNA'lar (circRNA'lar) kovalent olarak kapalı tek iplikçikli RNA'lar çoğu organizmada ifade edilir. Onlar bir downstream 5 'splice site, bir upstream 3' splice siteye katılarak oluşturulur, bir süreç geri birleştirme denir (Şekil 1A)1. 30-40 nt gibi kısa baz tamamlayıcı sergileyen pre-mRNA dizileri circRNA oluşumu2için uygun hizalama içine geri-splice siteleri getirmek . İnsanlarda, Alu elemanları1,genomunyaklaşık% 11 temsil 3 , kendi kendini tamamlayıcılık nedeniyle pre-mRNA geniş çift telli RNA yapıları oluşturmak4,5 ve böylece circRNAoluşumunuteşvik 1 .
Şu anda circRNA'ların üç ana işlevi tanımlanmıştır. Bazı circRNA'lar mikroRNA'ları (miRNA'lar) bağlar ve miRNA süngerleri gibi tutma hareketi ile6. CircRNA'lar transkripsiyonel ve post transkripsiyonel düzenlemeye, doğrusal birleştirme7 veya transkripsiyon faktörüaktivitesininmodülasyonu ile rekabet yoluyla 8. Son olarak, circRNA kısa açık okuma çerçeveleri ve ilke çalışmalarıkanıtı içeren 9,10tercüme edilebilir göstermektedir. Ancak, en circRNA fonksiyonu esrarengiz kalır. Dairesel RNA'ların çoğu yeni nesil sıralama yöntemleri kullanılarak tespit edilmiştir11. Hedeflenen RT-PCR yaklaşımları kullanılarak bireysel genlerin ayrıntılı analizleri, çok sayıda dairesel RNA'nın12.
Pre-mRNA işleme analiz etmek için muhabir genlerin kullanımı DNA muhabirinden elde edilen mRNA'nın hücrelere transfekte olan yapılarından elde edilen mRNA'nın analizi, dairesel RNA'lara uygulanabilen alternatif pre-mRNA birleştirme yi incelemek için kurulmuş bir yöntemdir. Genel olarak, alternatif ekson, çevresindeki intronlar ve kurucu ekonlar yükseltilir ve ökaryotik ifade vektörü içine klonlanır. Sık sık, intronlar kısalır. Yapılar ökaryotik hücrelere dönüştürüldü ve genellikle RT-PCR13,14tarafından analiz edilir. Bu yaklaşım, ortak transfeksiyon deneylerinde düzenleyici splice sitelerinin ve trans-etkili faktörlerin haritalarında yaygın olarak kullanılmıştır13,15,16,17,18. Buna ek olarak, protein ifade minigenlerin üretimi alternatif birleştirme değiştiren maddelerin taranması için izin19,20.
Yöntem dairesel RNA'lara uygulanmıştır. Literatürde en az 12 minigen omurgası tanımlanmış ve Tablo 1'deözetlenmiştir. TRNA tabanlı ifade sistemi21,22dışında hepsi polimeraz II organizatörleri bağlıdır. Burada, dairesel RNA'ların oluşumunda yer alan cis ve trans-acting faktörleri belirlemek için insan muhabir minigenleri oluşturmak için bir yöntem açıklıyoruz. Yayınlanan muhabir gen23 dizilerini kullanarak yönteme genel bir bakış Şekil 1'degösterilmiştir.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>(PEI) Hydrochloride</td>
			<td>Polysciences</td>
			<td>24765-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Builder tool</td>
			<td>NEB</td>
			<td></td>
			<td><a href="https://nebuilder.neb.com/#!/" target="_blank">https://nebuilder.neb.com/#!/</a></td>
		</tr>
		<tr>
			<td>Dark Reader Transilluminator.</td>
			<td>Clare Chemical Research</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Enzymatic DNA assembly kit</td>
			<td>NEB</td>
			<td>E2621S</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel and PCR cleanup kit</td>
			<td>Promega</td>
			<td>A9282</td>
			<td></td>
		</tr>
		<tr>
			<td>Glyco Blue</td>
			<td>Thermo Fisher</td>
			<td>AM9516</td>
			<td></td>
		</tr>
		<tr>
			<td>pcDNA3.1 cloning site</td>
			<td>Polycloning site</td>
			<td></td>
			<td><a href="https://assets.thermofisher.com/TFS-Assets/LSG/manuals/pcdna3_1_man.pdf" target="_blank">https://assets.thermofisher.com/TFS-Assets/LSG/manuals/pcdna3_1_man.pdf</a></td>
		</tr>
		<tr>
			<td>Polymerase 1</td>
			<td>NEB</td>
			<td>M0491L</td>
			<td>Q5 DNA polymerase</td>
		</tr>
		<tr>
			<td>Polymerase 2</td>
			<td>Biorad</td>
			<td>1725310</td>
			<td>Long range polymerase (NEB), iproof (BioRad)</td>
		</tr>
		<tr>
			<td>Polymerase 2</td>
			<td>Qiagen</td>
			<td>206402</td>
			<td>Qiagen long range polymerase kit</td>
		</tr>
		<tr>
			<td>Reverse Transcriptase</td>
			<td>Thermo Fisher</td>
			<td>18080044</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA isolation kit</td>
			<td>Life Technologies</td>
			<td>12183025</td>
			<td>Ambion by Life Technologies</td>
		</tr>
		<tr>
			<td>RNAse R</td>
			<td>Lucigen</td>
			<td>RNR07250</td>
			<td>Epicenter/Lucigen</td>
		</tr>
		<tr>
			<td>Stable competent cells</td>
			<td>NEB</td>
			<td>C3040H</td>
			<td>NEB stable cells</td>
		</tr>
		<tr>
			<td>Standard cloning bacteria</td>
			<td>NEB</td>
			<td>C2988J</td>
			<td>NEB5-alpha competent</td>
		</tr>
		<tr>
			<td>Web tool to design primers</td>
			<td>NEB</td>
			<td></td>
			<td><a href="https://nebuilder.neb.com/#!/" target="_blank">https://nebuilder.neb.com/#!/</a></td>
		</tr>
		<tr>
			<td>Web-based temperature calculations</td>
			<td>NEB</td>
			<td></td>
			<td><a href="https://tmcalculator.neb.com/#!/main" target="_blank">https://tmcalculator.neb.com/#!/main</a></td>
		</tr>
	</tbody>
</table>

use of alu element containing minigenes to analyze circular rnas

Lineer mRNA'lara ek olarak, birçok ökaryotik gen dairesel RNA'lar üretir. Çoğu dairesel RNA, 5'li bir splice sitesine, bir ön mRNA içinde 3' splice sitesi ile katılarak oluşturulur, bu süreç geri birleştirme olarak adlandırılır. Bu daireselleştirme büyük olasılıkla pre-mRNA ikincil yapılar tarafından desteklenir yakın içine splice siteleri getirmek. İnsan genlerinde, Alu elementleri bu ikincil RNA yapılarını teşvik eder, çünkü Alu elementleri bol miktarda bulunur ve mRNA öncesi zıt yönlerde mevcut olduğunda birbirleriyle baz tamamlayıcılıkları sergilerler. Burada dairesel RNA'lar oluşturan muhabir genleri içeren büyük Alu elementinin üretimini ve analizini tanımlıyoruz. Klonlama protokollerinin optimizasyonu ile 20 kb kesici uç uzunluğuna sahip muhabir genler oluşturulabilir. Ortak transfeksiyon deneylerindeki analizleri düzenleyici faktörlerin tanımlanmasına olanak sağlar. Böylece, bu yöntem dairesel RNA oluşumunda yer alan RNA dizilerini ve hücresel bileşenleri tanımlayabilir.

Muhabir genleri dairesel RNA oluşumunu etkileyen düzenleyici faktörlerin belirlenmesine olanak sağlar. Ancak, bu muhabir genler büyük tür ve genellikle DNA yapılarını kararsız hale getiren tekrarlayan elementler içerir. Büyük boyutları nedeniyle, genellikle eksonve daha küçük yan intronic parçalar içeren genomik parçaların yükseltilmesi ile elde edilen intronların bazı kısımlarını silmek gerekir. Bu DNA parçaları enzimatik olarak biraraya getirilerek konstrüksiyon enzimleri olmadan yapılm...

Watch this Scientific Journal Video about Use of Alu Element Containing Minigenes to Analyze Circular RNAs at JoVE.com

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

Dairesel RNA'lar üreten muhabir genleri klonlayıp analiz ediyoruz. Bu muhabir genler doğrusal birleştirme analiz etmek ve Alu elemanları içeren yapılar daha büyüktür. Dairesel RNA'ları araştırmak için yapılar hücrelere dönüştürülr ve doğrusal RNA çıkarıldıktan sonra RT-PCR kullanılarak ortaya çıkan RNA analiz edilir.

Dairesel RNA'ları Analiz Etmek Için Minigen İçeren Alu Elementin Kullanımı

In addition to linear mRNAs, many eukaryotic genes generate circular RNAs. Most circular RNAs are generated by joining a 5&#39; splice site with an ...

Bu protokol, kullanıcının alternatif birleştirmelerden geçebilen bir genomik ifade sistemi oluşturmasına olanak sağladığı ve bu durumda işlevleri ve hastalık ilişkisi için test edilebilen dairesel RNA'lar oluşturmasına olanak sağladığı için önemlidir. Bu tekniğin en büyük avantajı, dairesel RNA oluşumu nda ve işlevinde alternatif birleştirme eğitimi alırken başkalarının moleküler biyoloji ve klonlamada bu protokolü kullanmalarının önünü açacak olmasıdır. Birisi ilk kez bu tekniği deneyen benim tavsiyepcr koşullarını optimize etmek olacaktır.Degradeleri çalıştırın veya farklı tavlama sıcaklıkları ve uzatma süreleri ile touchdown PCR gerçekleştirin. Genellikle altı KB üzerinde uzun parçalar döngüsü başına bir KB uzatır ve farklı tavlama sıcaklıkları en iyi amplifikasyon için test edilmesi gerekir. Bu yöntemin gösterilmesi, kullanıcılara prosedürün özellikle astar ların neslinin daha zor yönlerini daha iyi görsel olarak göstereceği için önemlidir.Bu prosedürü başlatmak için UCSC Genom Tarayıcısını yükleyin ve dairesel RNA oluşumu için gerekli tekrarlayan elementleri belirlemek ve bunları yapılara dahil etmek için kullanın. Daha da önemlisi, amplifikasyon için astartekrarlayan elemanların dışında olması gerekir. Dairesel RNA sırasını insan BLAT aramasına yapıştırın ve doğru organizmayı seçin.Sırayı gönderin, tarayıcı görünümüne gidin ve 1,5 zamanlayıcıyı veya uygun şekilde uzaklaştırın. Ardından, kayan bir pencerede alt yazılarını tanımlamak için yinelenen öğelerin üzerinde fare. Alu elementler sinüs hattındadır.Yanlış bir görüntü elde edilirse tarayıcıyı sıfırlamak için pencerenin altındaki varsayılan parça düğmesini kullanın. İlk görüntülemek için gidin, USCS Genom Tarayıcıüst satırında DNA penceresinde gösterilen DNA dizisini indirmek için. Sıralama biçimlendirme seçeneğinde genişletilmiş büyük/renk seçeneklerini seçin.Varsayılan servis dosyasını daha düşük olarak seçin ve NCBI RefSeq için geçiş örneğini seçin. RepeatMasker için altı çizili, kalın ve Italik'i seçin. Gönder'i tıklatın.Büyük harfler, intronlar ise küçük harfler olarak ekonlar olacaktır. Ekson/intron sınırlarını kontrol edin. Tarayıcı ters tamamlayıcıyı gösteriyorsa, geri dön ve doğru ekson/intron kenarlıkları gösterene kadar ters tamamlayıcı kutusunu seçin.Ardından, dosyayı doğru yönlendirmeyle bir sözcük işleme belgesine kopyalayın ve ekseleri vurgulayın. Bu bölgelerdeki astarlar belirli dizileri yükseltmeyeceğinden, intron'un tekrarlayan bir bölgede başlayıp sona ermediğinden emin olmak için yükseltilecek parçaları seçin. Başlamak için, klonlama için astar tasarlamak için bir web aracı kullanın.Vektör dizisi için, ekleme bölgesini son nükleotit olarak ekleyin ve daha sonra parçaları ekleyin. Vektör numaralandırması belirli bir ekleme sitesiyle başlamadığından, vektöre ekleme yeri bulunur ve akış aşağı kısmı yukarı akış sırasının önüne konur. Erime noktaları dört santigrat dereceden fazlaysa ve amplifikasyonda çalışmıyorsa astarları ayarlayın.Bu çalışmada, dairesel RNA'lar üreten muhabir genler klonlanır ve analiz edilir. Optimize pcr ürünleri 1X jel yeşiliçeren% 1 agarose jel ayrılır. Tek tek bantlar enzimatik DNA montajında kullanılacak PCR ürünlerini temsil ediyor.Bu bantlar daha sonra jel kesilir ve saflaştırılır. Saflaştırılmış PCR ürün jel yeşili ile beklenen boyuta doğru çalışmaz, böylece ürünler de ürünlerin doğru boyutta olduğundan emin olmak için daha sonra ethidium bromür ile boyanmış bir % 1 agarose jel ayrılır. Başlamak için, enzimatik DNA montaj kiti ayarlayın.Vektör ve ekler bir ila iki molar oranında birleştirin. Daha sonra, 10 mikrolitre DNA montaj ana karışımı ekleyin. Örnekleri 50 derecede 60 dakika kuluçkaya yatırın.Kuluçka sırasında buz üzerindeki yetkili hücreleri eritin. Hücreler 50 mikrolitre lik bir hacimde olmalıdır. Ardından, toplam montaj reaksiyonu ile yetkili hücreleri dönüştürün.Yetkili hücrelere soğutulmuş monte ürünün iki mikrolitre ekleyin. Tüpü 4-5 kez hafifçe hareket ettirerek karıştırın. Girdap etmeyin.Karışımı 30 dakika buzüzerine yerleştirin. Isı 30 saniye boyunca 42 santigrat derecede karışımı şok. Sonra iki dakika buz üzerinde geri yerleştirin.Bundan sonra, tüpe oda sıcaklığında 950 mikrolitre SOC ortam ekleyin. 300 RPM'de sallayarak 60 dakika boyunca 37 santigrat derecede kuluçkaya yat. Bu kuluçka sırasında, uygun antibiyotik içeren sıcak iki seçim plakaları.Kuluçkadan sonra, reaksiyon tüpünü 10,000 g'de 30 saniye boyunca santrifüj edin ve bir seçim plakasındaki hücrelerin %25'ini, diğerinde %75'ini dışarı atın. Bu plakaları bir gecede 37 derecede kuluçkaya yatırın. Transfection başlamadan önce, kısıtlama sindirim dna kontrol edin.Burada, 9'dan 12'ye kadar ekson içeren temsili bir tau minigeni, majör rekombinasyonları ekarte etmek için gösterilen restriksiyon enzimleri ile kesilir. Transfeksiyon için, ilk pH iki mililitre başına bir miligram konsantrasyonda suda lineer polietilen hidroklorür çözünür. PH'ı yediye çıkarmak için sodyum hidroksit kullanın ve 0,22 mikrometre filtre ile çözeltiyi steril filtreleyin.Çözeltiyi kullanıma hazır olana kadar dört santigrat derecede saklayın. Daha sonra hücreleri altı kuyulu bir plakanın kuyularına bölün ve %10 FBS içeren DMEM ortamlarında bir gecede büyümelerine izin verin. Ertesi gün, steril bir tüp bildirilen genin bir mikrogram aliquot ve steril 150 milimoler sodyum klorür filtre steril 200 mikrolitre ekleyin.Girdap ile karıştırın. Daha sonra, DNA her bir mikrogram için polietileninin üç mikrolitre bir oranda bu karışıma polietileninin çözeltisi ekleyin. Tüpün alt kısmında örnekleri toplamak için kısaca santrifüj.Örnekleri oda sıcaklığında 10 dakika kuluçkaya yatırın. Daha sonra doğrudan HEK293 hücrelerine ekleyin. Hücreleri bir gecede %5 karbondioksitle 37 derecede kuluçkaya yatırın.Ertesi gün, RT-PCR için RNA'yı yalıtmak için bir RNA izolasyon kiti kullanın. insan beyin dokularından elde edilen iki örnekten cDNA dairesel RNA astarlar circ tau exon 1210 ters ve circ tau exon 1211 ileri ile yükseltilir. Tau dairesel RNA'ya karşılık gelen beklenen bant görülürken, diğer güçlü bantlar insan genomuna uymayan eserlerdir.Bu deney aynı PCR koşulları ile tekrarlanır, ancak ters transkripsiyon sadece circ tau exon 1210 ters astar ile gerçekleştirilmiştir. Yalnızca beklenen bant yükseltilir ve sıralama ile doğrulanır. RNA daha sonra doğrusal RNA'yı kaldıran RNa R ile tedavi edilir.Dairesel RNA tedaviden sonra saptanabilirken, lineer RNA artık tespit edilebilir bir sinyal vermez. RNA transfeksiyon sonrası 24 saat izole edilir ve RT-PCR tarafından analiz edilir. Lineer tau mRNA amplifikasyonu ekson 10'un alternatif birleştirilmesi nedeniyle iki bant gösterir.Onların oranı birleştirme faktörlerinin aşırı ifade değişir. Dairesel 1210 tau RNA amplifikasyon bazı birleştirme faktörleriözellikle Cdc2 gibi kinaz CLK2 ve SR protein 9G8 ifade tau circ RNA ifade bağımlılığı gösterir. Bu yordamı çalışırken hatırlanması gereken en önemli şey, bir minigen inşa ederken astarların tasarımı ve konumudur.Astar tekrarlayan elemanlar içinde olmamalıdır ve yükseltilen parçaya bağlı olarak farklı koşullar altında optimize edilmesi gerekir. Bu yordamı takiben, uygulanabilecek diğer yöntemler dairesel RNA'ların işlevini test edilecektir. Kullanıcılar, kullanıcının ilgilendiği belirli dairesel RNA'nın işlevini belirlemek için proteinlerin çevirisini veya haczini test edebilirler.Bu teknik, genomik parçaların minigen sisteminde kullanılmasının yolunu açarak, kullanıcının işlevlerini test edip tanımlamasına ve bazı yönlerden hastalıkla ilişkisini test etmesine ve tanımlamasına olanak tanıyan dairesel RNA'ları ifade edebilir. Bu tekniğin anlamı potansiyel tedaviler ve belirli bir hastalığın tanısı doğru uzanır, örneğin tauopapatiler, tau patolojisi ile ilişkili nörodejeneratif hastalıklar. MAPT olarak bilinen mikrotübül ilişkili protein tau'nun, hastalık patolojisine katkıda bulunduğuna inandığımız dairesel RNA'lar ürettiğini keşfettik ve bu yöntem bu dairesel RNA'ları tam olarak incelememize ve hastalıkla olan işlevlerini ve ilişkilerini belirlememize olanak sağlıyor.Bu yöntem, dairesel RNA'ların daha iyi anlaşılması, işlevlerinin ne olduğu ve bazı hastalıklarda oynayabilecekleri rolün daha iyi anlaşılmasını sağlayabilir. Bu yöntem, türler arasında dairesel RNA'ları ifade eden diğer minigenlerin klonlanmasında uygulanabilir ve bu yöntem işlevleri hakkında bilgi verebilir. PCR ürünlerinin amplifikasyonu, genomik DNA'dan güçlendirilmiş uzun parçalar ve parça içinde birden fazla tekrarlayan eleman olması yla en zor uymuş.

use-of-alu-element-containing-minigenes-to-analyze-circular-rnas

Research

JoVE Journal

Biochemistry

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

Combining Wet and Dry Lab Techniques to Guide the Crystallization of Large Coiled-coil Containing Proteins

Obtaining crystals for structure determination can be a difficult and time consuming proposition for any protein. Coiled-coil proteins and domains are found throughout nature, however, because of their physical properties and tendency to aggregate, they are traditionally viewed as being especially difficult to crystallize. Here, we utilize a variety of quick and simple techniques designed to identify a series of possible domain boundaries for a given coiled-coil protein, and then quickly characterize the behavior of these proteins in solution. With the addition of a strongly fluorescent tag (mRuby2), protein characterization is simple and straightforward. The target protein can be readily visualized under normal lighting and can be quantified with the use of an appropriate imager. The goal is to quickly identify candidates that can be removed from the crystallization pipeline because they are unlikely to succeed, affording more time for the best candidates and fewer funds expended on proteins that do not produce crystals. This process can be iterated to incorporate information gained from initial screening efforts, can be adapted for high-throughput expression and purification procedures, and is augmented by robotic screening for crystallization.

We describe a framework incorporating straightforward biochemical and computational analysis to guide the characterization and crystallization of large coiled-coil domains. This framework can be adapted for globular proteins or extended to incorporate a variety of high-throughput techniques.

Islak ve kuru Lab Teknikleri birleştiren Büyük Spiral-coil İçeren Proteinler kristalleşmesini Kılavuzu

Obtaining crystals for structure determination can be a difficult and time consuming proposition for any protein. Coiled-coil proteins and domains are ...

Biyokimya

Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the availability of enough area to perform the function. Nanodiscs are used to provide a membrane surface of controlled size and lipid content. In the absence of bound extrinsic proteins, sodium phosphotungstate-stained nanodiscs appear as stacks of coins when viewed from the side by transmission electron microscopy (TEM). This protocol is therefore designed to intentionally promote stacking; consequently, the prevention of stacking can be interpreted as the binding of the membrane-binding protein to the nanodisc. In a further step, the TEM images of the protein-nanodisc complexes can be processed with standard single-particle methods to yield low-resolution structures as a basis for higher resolution cryoEM work. Furthermore, the nanodiscs provide samples suitable for either TEM or non-denaturing gel electrophoresis. To illustrate the method, Ca2+-induced binding of 5-lipoxygenase on nanodiscs is presented.

Many proteins perform their function when attached to membrane surfaces. The binding of extrinsic proteins on nanodisc membranes can be indirectly imaged by transmission electron microscopy. We show that the characteristic stacking (rouleau) of nanodiscs induced by the negative stain sodium phosphotungstate is prevented by the binding of extrinsic protein.

Yöntem gözünüzde canlandırın ve Transmisyon Elektron Mikroskobu Membran Etkileşen Proteinler Analiz için

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the ...

Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

Transfer RNA (tRNA) is an essential part of the translational machinery in any organism. tRNAs bind and transfer amino acids to the translating ribosome. The relative levels of different tRNAs, and the ratio of aminoacylated tRNA to total tRNA, known as the charging level, are important factors in determining the accuracy and speed of translation. Therefore, the abundance and charging levels of tRNAs are important variables to measure when studying protein synthesis, for example under various stress conditions. Here, we describe a method for harvesting tRNA and directly measuring both the relative abundance and the absolute charging level of specific tRNA species in Escherichia coli. The tRNA is harvested in such a way that the labile bond between the tRNA and its amino acid is preserved. The RNA is then subjected to gel electrophoresis and Northern blotting, which results in separation of the charged and uncharged tRNAs. The levels of specific tRNAs in different samples can be compared due to the addition of spike-in cells for normalization. Prior to RNA purification, we add 5% of E. coli cells that overproduce the rare tRNAselC to each sample. The amount of the tRNA species of interest in a sample is then normalized to the amount of tRNAselC in the same sample. Addition of spike-in cells prior to RNA purification has the advantage over addition of purified spike-in RNAs that it also accounts for any differences in cell lysis efficiency between samples.

Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

Here we present a method for directly measuring transfer RNA charging levels from purified Escherichia coli RNA as well as a way to compare relative levels of transfer RNA, or any other short RNA, across different samples based on the addition of spike-in cells expressing a reference gene.

Miktar bolluk ve Escherichia coli olarak Transfer RNA'ların düzeyde şarj

Transfer RNA (tRNA) is an essential part of the translational machinery in any organism. tRNAs bind and transfer amino acids to the translating ribosome. ...

Simultaneous Measurement of Superoxide/Hydrogen Peroxide and NADH Production by Flavin-containing Mitochondrial Dehydrogenases

It has been reported that mitochondria can contain up to 12 enzymatic sources of reactive oxygen species (ROS). A majority of these sites include flavin-dependent respiratory complexes and dehydrogenases that produce a mixture of superoxide (O2&#9679;-) and hydrogen peroxide (H2O2). Accurate quantification of the ROS-producing potential of individual sites in isolated mitochondria can be challenging due to the presence of antioxidant defense systems&#160;and side reactions that also form O2&#9679;-/H2O2. Use&#160;of nonspecific inhibitors that can disrupt mitochondrial bioenergetics can also compromise measurements by altering&#160;ROS release from other sites of production. Here, we present an easy method for the simultaneous measurement of H2O2 release and nicotinamide adenine dinucleotide (NADH) production by purified flavin-linked dehydrogenases. For our purposes here, we have used purified pyruvate dehydrogenase complex (PDHC) and &#945;-ketoglutarate dehydrogenase complex (KGDHC) of porcine heart origin as examples. This method allows for an accurate measure of native H2O2 release rates by individual sites of production by eliminating other potential sources of ROS and antioxidant systems. In addition, this method allows for a direct comparison of the relationship between H2O2 release and enzyme activity and the screening of the effectiveness and selectivity of inhibitors for ROS production. Overall, this approach can allow for the in-depth assessment of native rates of ROS release for individual enzymes prior to conducting more sophisticated experiments with isolated mitochondria or permeabilized muscle fiber.

Mitochondria contain several flavin-dependent enzymes that can produce reactive oxygen species (ROS). Monitoring ROS release from individual sites in mitochondria is challenging due to unwanted side reactions. We present an easy, inexpensive method for direct assessment of native rates for ROS release using purified flavoenzymes and microplate fluorometry.

Süperoksit/hidrojen peroksit ve NADH üretim mitokondriyal Dehydrogenases Flavin içeren tarafından eş zamanlı ölçüm

It has been reported that mitochondria can contain up to 12 enzymatic sources of reactive oxygen species (ROS). A majority of these sites include ...

Use of Two Dimensional Semi-denaturing Detergent Agarose Gel Electrophoresis to Confirm Size Heterogeneity of Amyloid or Amyloid-like Fibers

Amyloid or amyloid-like fibers have been associated with many human diseases, and are now being discovered to be important for many signaling pathways. The ability to readily detect the formation of these fibers under various experimental conditions is essential for understanding their potential function. Many methods have been used to detect the fibers, but not without some drawbacks. For example, electron microscopy (EM), or staining with Congo Red or Thioflavin T often requires purification of the fibers. On the other hand, semi-denaturing detergent agarose gel electrophoresis (SDD-AGE) allows detection of the SDS-resistant amyloid-like fibers in the cell extracts without purification. In addition, it allows the comparison of the size difference of the fibers. More importantly, it can be used to identify specific proteins within the fibers by Western blotting. It is less time consuming and more easily accessible to a wider number of labs. SDD-AGE results often show variable degree of heterogeneity. It raises the question whether part of the heterogeneity results from the dissociation of the protein complex during the electrophoresis in the presence of SDS. For this reason, we have employed a second dimension of SDD-AGE to determine if the size heterogeneity seen in SDD-AGE is truly a result of fiber heterogeneity in vivo and not a result of either degradation or dissociation of some of the proteins during electrophoresis. This method allows fast, qualitative confirmation that the amyloid or amyloid-like fibers are not partially dissociating during the SDD-AGE process.

Here we use two-dimensional semi-denaturing agarose gel electrophoresis to confirm the presence of amyloid-like fibers of heterogeneous size and exclude the possibility that the size heterogeneity is due to dissociation of the amyloid fibers during the gel running process.

İki boyutlu yarı denaturing deterjan özel Jel Elektroforez amiloid veya amiloid benzeri liflerin boyutu heterojen onaylamak için kullanımı

Amyloid or amyloid-like fibers have been associated with many human diseases, and are now being discovered to be important for many signaling pathways. ...

Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli

With increasing interest in RNA biology and the use of RNA molecules in sophisticated biotechnological applications, the methods to produce large amounts of recombinant RNAs are limited. Here, we describe a protocol to produce large amounts of recombinant RNA in Escherichia coli based on co-expression of a chimeric molecule that contains the RNA of interest in a viroid scaffold and a plant tRNA ligase. Viroids are relatively small, non-coding, highly base-paired circular RNAs that are infectious to higher plants. The host plant tRNA ligase is an enzyme recruited by viroids that belong to the family Avsunviroidae, such as Eggplant latent viroid (ELVd), to mediate RNA circularization during viroid replication. Although ELVd does not replicate in E. coli, an ELVd precursor is efficiently transcribed by the E. coli RNA polymerase and processed by the embedded hammerhead ribozymes in bacterial cells, and the resulting monomers are circularized by the co-expressed tRNA ligase reaching a remarkable concentration. The insertion of an RNA of interest into the ELVd scaffold enables the production of tens of milligrams of the recombinant RNA per liter of bacterial culture in regular laboratory conditions. A main fraction of the RNA product is circular, a feature that facilitates the purification of the recombinant RNA to virtual homogeneity. In this protocol, a complementary DNA (cDNA) corresponding to the RNA of interest is inserted in a particular position of the ELVd cDNA in an expression plasmid that is used, along the plasmid to co-express eggplant tRNA ligase, to transform E. coli. Co-expression of both molecules under the control of strong constitutive promoters leads to production of large amounts of the recombinant RNA. The recombinant RNA can be extracted from the bacterial cells and separated from the bulk of bacterial RNAs taking advantage of its circularity.

Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli

Here, we present a protocol to produce large amounts of recombinant RNA in Escherichia coli by co-expressing a chimeric RNA that contains the RNA of interest in a viroid scaffold and a plant tRNA ligase. The main product is a circular molecule that facilitates purification to homogeneity.

Escherichia coli içinde Viroid kaynaklı bir sistemi kullanarak dairesel bir iskele üzerinde rekombinant RNA'ların büyük ölçekli üretim

With increasing interest in RNA biology and the use of RNA molecules in sophisticated biotechnological applications, the methods to produce large amounts ...

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this superfamily generally display multi-functionality, involving mainly hydrolase and decarboxylase mechanisms. This article presents a series of consecutive methods for the expression and purification of FAHD proteins, mainly FAHD protein 1 (FAHD1) orthologues among species (human, mouse, nematodes, plants, etc.). Covered methods are protein expression in E. coli, affinity chromatography, ion exchange chromatography, preparative and analytical gel filtration, crystallization, X-ray diffraction, and photometric assays. Concentrated protein of high levels of purity (&#62;98%) may be employed for crystallization or antibody production. Proteins of similar or lower quality may be employed in enzyme assays or used as antigens in detection systems (Western-Blot, ELISA). In the discussion of this work, the identified enzymatic mechanisms of FAHD1 are outlined to describe its hydrolase and decarboxylase bi-functionality in more detail.

Expression and purification of fumarylacetoacetate hydrolase domain-containing proteins is described with examples (expression in E. coli, FPLC). Purified proteins are used for crystallization and antibody production and employed for enzyme assays. Selected photometric assays are presented to display the multi-functionality of FAHD1 as oxaloacetate decarboxylase and acylpyruvate hydrolase.

Fumarylacetoasetat Hydrolase etki alanı Içeren proteinleri ifade, arıtma, kristalizasyon ve enzim Asdimleri

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this ...

Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane trafficking, signal transduction and cytoskeletal remodeling. Classic tools for measuring such interactions include surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). While powerful tools, these approaches have setbacks. ITC requires large amounts of purified protein as well as lipids, which can be costly and difficult to produce. Furthermore, ITC as well as SPR are very time consuming, which could add significantly to the cost of performing these experiments. One way to bypass these restrictions is to use the relatively new technique of microscale thermophoresis (MST). MST is fast and cost effective using small amounts of sample to obtain a saturation curve for a given binding event. There currently are two types of MST systems available. One type of MST requires labeling with a fluorophore in the blue or red spectrum. The second system relies on the intrinsic fluorescence of aromatic amino acids in the UV range. Both systems detect the movement of molecules in response to localized induction of heat from an infrared laser. Each approach has its advantages and disadvantages. Label-free MST can use untagged native proteins; however, many analytes, including pharmaceuticals, fluoresce in the UV range, which can interfere with determination of accurate KD values. In comparison, labeled MST allows for a greater diversity of measurable pairwise interactions utilizing fluorescently labeled probes attached to ligands with measurable absorbances in the visible range as opposed to UV, limiting the potential for interfering signals from analytes.

Microscale thermophoresis obtains binding constants quickly at low material cost. Either labeled or label free microscale thermophoresis is commercially available; however, label free thermophoresis is not capable of the diversity of interaction measurements that can be performed using fluorescent labels. We provide a protocol for labeled thermophoresis measurements.

Protein-Lipid Etkileşimlerini Ölçmek için Mikro Ölçekli Termoforez Kullanımı

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane ...

Ready-To-Use qPCR for Detection of DNA from Trypanosoma cruzi or Other Pathogenic Organisms

Real-time PCR (qPCR) is a remarkably sensitive and precise technique&#160;that allows for amplifying&#160;minute amounts of nucleic acid targets from a multitude of samples. It has been extensively used in many research areas and achieved industrial application in fields such as human diagnostics and trait selection in crops of genetically modified organisms (GMO) crops. However, qPCR is not an error-proof technique. Mixing all reagents into a single master mix subsequently distributed onto 96 wells of a regular qPCR plate might lead to operator mistakes such as incorrect mixing of reagents or inaccurate dispensing into the wells. Here, a technique called gelification is presented, whereby most of the water present in the master mix is substituted by reagents that form a sol-gel mixture when submitted to a vacuum. As a result, qPCR reagents are effectively preserved for a few weeks at room temperature or a few months at 2-8 oC. Details of preparing each solution are shown here along with the expected aspect of a gelified reaction designed to detect&#160;T. cruzi satellite DNA (satDNA). A similar procedure can be applied to detect other organisms. Starting a gelified qPCR run is as simple as removing the plate from the refrigerator, adding the samples to their respective wells, and starting the run, thus decreasing the setup time of a full-plate reaction to the time it takes to load the samples. Additionally, gelified PCR reactions can be produced and controlled for quality in batches, saving time and avoiding common operator mistakes while running routine PCR reactions.

Ready-To-Use qPCR for Detection of DNA from Trypanosoma cruzi or Other Pathogenic Organisms

The present work describes the steps for producing ready-to-use qPCR for T. cruzi DNA detection that can be pre-loaded on the reaction vessel and stored in the refrigerator for several months.

Trypanosoma cruzi veya Diğer Patojenik Organizmalardan DNA Tespiti için Kullanıma Hazır qPCR

Real-time PCR (qPCR) is a remarkably sensitive and precise technique&#160;that allows for amplifying&#160;minute amounts of nucleic acid targets from a ...

Formulation and Characterization of Bioactive Agent Containing Nanodisks

The term nanodisk refers to a discrete type of nanoparticle comprised of a bilayer forming lipid, a scaffold protein, and an integrated bioactive agent. Nanodisks are organized as a disk-shaped lipid bilayer whose perimeter is circumscribed by the scaffold protein, usually a member of the exchangeable apolipoprotein family. Numerous hydrophobic bioactive agents have been efficiently solubilized in nanodisks by their integration into the hydrophobic milieu of the particle&#39;s lipid bilayer, yielding a largely homogenous population of particles in the range of 10-20 nm in diameter. The formulation of nanodisks requires a precise ratio of individual components, an appropriate sequential addition of each component, followed by bath sonication of the formulation mixture. The amphipathic scaffold protein spontaneously contacts and reorganizes the dispersed bilayer forming lipid/bioactive agent mixture to form a discrete, homogeneous population of nanodisk&#160;particles. During this process, the reaction mixture transitions from an opaque, turbid appearance to a clarified sample that, when fully optimized, yields no precipitate upon centrifugation. Characterization studies involve the determination of bioactive agent solubilization efficiency, electron microscopy, gel filtration chromatography, ultraviolet visible (UV/Vis) absorbance spectroscopy, and/or fluorescence spectroscopy. This is normally followed by an investigation of biological activity using cultured cells or mice. In the case of nanodisks harboring an antibiotic (i.e., the macrolide polyene antibiotic amphotericin B), their ability to inhibit the growth of yeast or fungi as a function of concentration or time can be measured. The relative ease of formulation, versatility with respect to component parts, nanoscale particle size, inherent stability, and aqueous solubility permits myriad in vitro and in vivo applications of nanodisk technology. In the present article, we describe a general methodology to formulate and characterize nanodisks containing amphotericin&#160;B as the hydrophobic bioactive agent.

Here, we describe the production and characterization of bioactive agents containing nanodisks. Amphotericin B nanodisks are taken as an example to describe the protocol in a stepwise manner.

Nanodisk İçeren Biyoaktif Ajanın Formülasyonu ve Karakterizasyonu

The term nanodisk refers to a discrete type of nanoparticle comprised of a bilayer forming lipid, a scaffold protein, and an integrated bioactive agent. ...