Bu çalışma Sağlık RO1-GM085130 Ulusal Enstitüsü ve Ulusal Bilim Vakfı MCB0929394 bağışları ile destek verdi. Dr. Marion Schmidt Biz onun misafirperverliği ve onun laboratuar film bize izin verdiği için teşekkür ediyorum.

1. Footprinting Reaktifler hazırlanması <ol><li> 100 mM sodyum cacodylate, 1 mM EDTA ve 1 M KCl içeren bir 10x reaksiyon tamponu hazırlayın. PH 7.4 &#39;e ayarlayın. Filtre 0.2 mcM asetat filtre cihazı (Nalgene) kullanarak tampon. Not: 10x tampon doğrudan pipetlemeyin RNA yok. </li><li> Tablo 1&#39;de gösterildiği gibi her bir reaksiyon için titrasyon reaksiyon karışımı hazırlayın. Titrasyon karışımı hacmi (1x istenilen konsantrasyonda tampon ve Mg (II)), 1x tampon RNA 10μl eklemeden önce, 90 ul olmalıdır. </li><li> 6.63M Üre, 20mm sodyum sitrat, 1mm EDTA, 0,25 mg / ml tRNA,% 0.025 ksilen cyanol,% 0.025 bromphenol mavi içeren bir RNAse T1 sindirim tampon hazırlayın. Bu tampon, 6 aya kadar 4 ° C saklanabilir. </li><li> Fenton reaksiyonu 250 mM Fe (NH 4) 2 (SO 4) 2, 250 mM EDTA ve 500 mM sodyum L-askorbat taze olarak hazırlanan sulu çözeltiler gerektirir . Konsantrasyonları bölüm 1 not0,5 radikaller ve çeşitli kalsiyum iyon yoğunluğunun değil scavenge sodyum cacodylate gibi tampon denge deneyleri için en uygun olan. </li><li> Peroksidatif (1.5.1) ve oksidatif (1.5.2) footprinting reaksiyon için Reaktifler. <ol><li> Peroksidatif Fenton reaksiyonu 100 mM Fe-EDTA çözeltisi ve 50 mM sodyum L-askorbat ve% 1.5 H 2 O 2 taze olarak hazırlanan sulu çözeltiler gerektirir . Fe-EDTA çözeltisi, demir üzerinde EDTA 1.1 kat fazla karıştırma Fe (NH 4) 2 (SO 4) 2 EDTA ile hazırlanmıştır . </li><li> Sadece oksidatif parçalanma reaksiyonu (3.4.2) başlamadan önce, 2 ul ul Fe (NH 4) 2 (SO 4) 2 (250 mM), 2.2 EDTA ul (250 mM), 62.5 birleştirerek Fenton reaksiyon karışımı oluşturmak sodyum L-askorbat (500 mM), 22.5 ul 10x reaksiyon tamponu ve 0 footprinting reaksiyon karışımı (3.4.2) nihai konsantrasyonlarını ulaşmak için H 2 O 135,5 ul.10 mM 0.11 mM ve 6.61 mM, sırasıyla. </li></ol></li></ol> 2. RNA footprinting Denemeye Hazırlık <ol><li> 50 nükleotidler, daha kısa (Integrated DNA Teknolojileri, www.idtdna.com gibi) satın alınması, RNA, DNA şablonları 6 in vitro transkripsiyon standart tarafından üretilen ya da olabilir . Footprinting deneyler sırasında RNA örnek bütünlüğünü korumak için 7 tavsiye edilen laboratuvar temizlik prosedürlerini izleyin . </li><li> Defosforilasyon 8 in vitro transkripsiyonu RNA önce RNA&#39;nın 5 &#39;son 32 P etiketleme için gerekli. RNA 10 pmol kinasing tarafından uç-etiketlenmiş 9 çözüm fosfataz başarılı inaktivasyon sonra bulutlu olmalıdır. </li><li> Denatüre jel elektroforezi kullanılarak radyoaktif etiketli RNA arındırın. RNA 0.3 M sodyum asetat (pH 5.5) ile iki sonraki jel ekstraksiyon tarafından kurtarılır. RNA ile 0,5 ml 1 ml etanol ve sonraki sulu çözeltisi karıştırılarak çöktürülmüşkuru buz üzerinde 1 dakika inkübasyon. 4 ° C, 0,5 saat süreyle 12.500 rpm örnek Spin. Süpernatantı atın ve RNA pelet% 70 etanol ile yıkayın. Ek santrifüj sonra süpernatantı ve pelet kuru vakum. </li><li> 1x bir reaksiyon tüpünde RNA çözümü, etanol ve vakumlu kuru RNA ile çökelti Buffer.Pipet 30 ul 330 ul 32 P etiketli RNA örnekleri çözülür; referans merdiveni (adım 3.5) üretmek için bu tüp tutmak. 95 ısıtarak tamponlu RNA çözüm ° C &#39;de 2 dakika süreyle denatüre. Örnekleri 15 dakika oda sıcaklığında soğutun ve çözelti içinde tüplerin kapakları kondanseleri getirmek için hızlı bir spin vermek. </li></ol> 3. Footprinting Deney <ol><li> Elektroforez tarak, veri noktalarının analitik jel başına maksimum sayısını belirler. Biz 30 iyi bir tarak kullanın. Referans merdiven, 2 kuyu ve uncleaved kontrolü birini kaplar. 27 reaksiyon tüpleri hazırlayın. Son Mg (II) concentratititrasyon orta noktası etrafında büyüklük emirleri kapsayan bir logaritmik ölçekte ons eşit aralıklı olmalıdır. </li><li> Ayrı ayrı 50 RNA ve Mg (II) çözeltileri ° C, 5 dakika boyunca inkübe edin. 100 ul son bir hacme ulaşmak için 1x tampon karşılık gelen miktarda Mg (II) 90 ul ile 10 ul RNA çözüm karıştırın. 50 - 30 dakika çözümler inkübe ° C </li><li> Katlama oluşur sırasında çözümleri, 25 ° C&#39;de 1 saat için muvazene edelim. </li><li> Peroksidatif (3.4.1) veya oksidatif (3.4.2) hidroksil radikal footprinting reaksiyon. <ol><li> 2 ul Fe-EDTA (100 mM) damlacıkları koyarak peroksidatif reaksiyonu başlatın, 2 ul sodyum L-askorbat (50 mM) ve 2 ul H 2 O 2 (% 1.5) reaksiyon üst içinde birbirinden ayrılmış tüp RNA çözüm içeren ve dinç karıştırılarak footprinting reaksiyon başlatabilir. 300 ul soğuk mutlak etanol ekleyerek 15 saniye sonra tepki durdurun. Tüpler 3-5 kez çevirin. Çökelme, yıkama veadım 2.4 olarak pelet kurulayın. </li><li> Oksidatif hidroksil radikal footprintingreaction taze hazırlanmış Fenton reaksiyon karışımı (1.6) 5 ul ekleyerek başlayın. 30 dakika boyunca inkübe 25 ° C Tüpler 3-5 kez çevirerek tepki ve karıştırın gidermek için soğuk mutlak etanol 300 ul ekleyin. Çökelme, yıkama ve kuru pelet gibi adım 2.4 &#39;de belirtilen. </li></ol></li><li> Adım 1.3 &#39;te hazırlanan çözüm kullanarak standart prosedürler 10 göre bir RNaz T1 sindirmek referans örnek oluşturun. </li><li> 8 ul jel yükleme boyası II (Ambion) ve onaylamak RNA RNA pelet çözülür Geiger sayacı kullanarak yeniden süspanse. </li><li> 11 Yük örnekleri iki referans ve kontrol dahil olmak üzere standart protokollere göre denatüre,% 8&#39;lik poliakrilamid sıralama jel hazırlayın. 60 W Ayrı RNA parçaları - 2.5 saat için 75 W. Gecede bir depolama fosfor ekrana kurutulmuş jel Açığa. Tarama fosfor filmsiz otoradyografi, örneğin bir görüntüleme sistemi ile ekran. Fırtına 865 (GE Healthcare) veya Typhoon (GE Healthcare). Jel görüntü dosyası, bilgisayarınıza aktarın. </li></ol> 4. Veri Analizi <ol><li> Her bir nükleotid hidroksil radikal reaktivite analitik jel her bandın şiddeti nicel tarafından belirlenir. Karşıdan yükleyin, kurun ve açık SAFA, tek bant montaj ve ölçümü için açık kaynak kodlu yazılım kullanımı kolay bir 12, 13 kullanım kılavuzu yönergeleri izleyin. Kısaca, RNA dizisi olarak yükleyin. Txt dosyası. Jel dosyası olarak jel resmi izledi. Bant yoğunlukları ayarlayın, şerit tanımlamak, bir çapa şeritli seçin ve bir jel hizalama. Nükleotidleri Atama RNaz T1 sindirim merdiven referans gruplarından oluşur. Grubun integrallerin sayısal olarak ve potansiyel değişmez artıkları normalleştirmek ve atamak için normalleşme / colorplot özelliğini kullanabilirsiniz. Çıktı. Txt dosyası olarak kaydedin. </li><li> SAFA temsil jel üzerinde şerit ve satır temsil eden sütunlar içeren bir elektronik tablo çıktılarıRNA parçası karşılık gelen bireysel bantları bant yoğunluğu tegrated. İlk olarak, çözücü erişilebilirlik gözle görülür bir değişim profili ile bitiş noktası Mg (II) konsantrasyonu (bizim örneğimizde 50 mM) Mg (II) örnek türetilmiş koruma profilinin karşılaştırarak ekran koruma siteleri de belirleyebilir. Değer ne kadar düşükse, daha nükleotid hidroksil radikalleri ve tersi saldırıya karşı korunmaktadır. </li><li> Mg (II) konsantrasyonu karşı nükleotidler bireysel ya da gruplar normalize bant integraller katlanır izotermleri oluşturun. • - izotermleri fi = L + (L U) tarafından ayrı ayrı kesirli doygunluk ölçeklendirilir <img src="files/ftp_upload/3244/3244y.jpg" alt="Y" /> f grup (lar) entegre yoğunluğu gösterir analiz edilen L ve U geçiş için alt ve üst sınırları temsil eden ve <img src="/files/ftp_upload/3244/3244y.jpg" alt="Y" /> kesirli doygunluk 14 verilerin sığması non-lineer l kullanarakdoğu-Hill denklemi (1) kare analiz programı (ya Kökeni (OriginLab) veya GraphPad (GraphPad Software, Inc.) kullanın.) <img alt="Denklem 1" src="/files/ftp_upload/3244/3244eq1.jpg" /> K d denge olduğu asitlik sabiti, [M] katlanır tepki aracılık değişken, bu örnekte Mg (II) konsantrasyonu gelir ve n H Hill katsayısı. Bu prosedür, kesirli doygunluk geçiş terazi, orta noktası belirler ve fenomenolojik bir geçiş sigmoidal olup olmadığını test sağlar. SAFA (Şekil 3B) tarafından analiz edilir ve yukarıda açıklandığı gibi uygun sıralama jeller (Şekil 3A) türetilen izotermleri Şekil 3C gösterilmiştir. Şekil 3&#39;te gösterildiği gibi kesirli doygunluk izotermleri bir elektronik tablo ölçeklendirme tarafından oluşturulan denklem fraksiyonu kullanarak en uygun alt ve üst sınırları (U ve L) karşı SAFA tarafından oluşturulan değerleri = değer / (U-L) - L / (U - L). </li></ol> 5. Temsilcisi Sonuçlar: Şekil 3, P4-P6 RNA • OH footprinting deneyler temsilcisi sonuçlar gösterir. Jel görüntüsünde (solda) T1 şeritli (Her G RNaz T1 parçalayarak) iyi tanımlanmış bantları nedeniyle olası tek nükleotid atama)) arka plan bölünme, sağ şeritte (b) az olduğunu gösterir, ve c) hidroksil radikali RNA indüklenmiş parçalanma iyi bir arka plan üzerindedir. RNA üçüncül yapısının oluşumu gösteren bantları bireysel ve gruplar entegre bant yoğunluğu azalan düşük ve yüksek Mg (II) geçiş bağlanmıştır. Tek veya bölünme değişiklikler eş zamanlı olarak &#39;koruma&#39; olarak adlandırılır bitişik nükleotid grupları. Mg (II) yakından kristal yapısında gözlenen molekülünün çözücü ulaşılmaz bölgelere karşılık gelen karakteristik • OH korumaları P4-P6 RNA katlama aracılı 15 Katlanmış RNA molekülleri c<html> bir çok farklı açık, yapısal ya da dinamik bir fenomen ile ilişkili olmamıştır üçüncül kişi koruma kapsamlarını (yani, nasıl arka bant yoğunlukları yakın düşüş) var. Böylece, bazı RNA molekülleri Şekil 3&#39;te gösterildiği gibi, iyi tanımlanmış yapısal geçişler göstermek, ve bazı olmayacaktır. Band şiddetleri SAFA 12 analizi (Şekil 3B) tarafından sayısal ve normalize edilir. Çıkış • OH karşı koruma göreceli derecesi görselleştirmek bir &#39;termal&#39; komplo. Renk geçişi, Mg (II) Ayrıca üzerine erişilebilirlik değişikliği açıklamaktadır. Sırasıyla kırmızı veya mavi, daha erişilebilir ve daha korumalı nükleotidler, Beyaz. Gölgeleme her bir derece, bir koruma eğrisi (Şekil 3C) olarak çizilen ve Hill denklemi (1) gibi bağlayıcı bir model ile analiz edilebilir bir sayısal değeri ile bağlanmıştır. Sabit disosiasyon Koruma 153-155 bağlı denge Koruma 163-164 karşılık gelen değeri yaklaşık iki kat fazladır. ontent &quot;&gt; <img alt="Şekil 1" src="/files/ftp_upload/3244/3244fig1.jpg" /> Şekil 1 Hidroksil radikal üretimi ve P4-P6 RNA katlanır. A) Fe (II), hidrojen peroksit, hidroksil radikalleri üretimi katalize eder. Askorbat demir demir Fe (III) geri azaltır. B) Mg (II) yokluğunda, hiçbir P4-P6 üçüncül yapısı, hidroksil radikalleri erişilebilir tüm omurga pozisyonları nüfuz ve tutunmaya izin oluşur. Mg (II) eklenmesi, sadece solvent erişilebilir omurga hidroksil radikalleri tarafından bölünmüş izin, P4-P6 katlayarak başlatır. <img alt="Şekil 2" src="/files/ftp_upload/3244/3244fig2.jpg" /> Şekil 2 hidroksil radikal footprinting deney için Anahat. A) Dephophorylation ve RNA 32 P 5&#39;-etiketleme. B) 32 Arıtma denatüre poliakrilamid jel RNA P-etiketli. C) RNA bant eksizyonu sonraki RNA ekstraksiyonu ve etanol yağış. D) Prefolding veRNA katlanır. E) taze hazırlanmış Fenton reaksiyon karışımının eklenmesi hidroksil radikalleri oluşturur. F) poliakrilamid jel elektroforezi denatüre RNA parçası ayırma. G) SAFA yazılımı ile RNA parçaları kantitasyonu. <img alt="Şekil 3" src="/files/ftp_upload/3244/3244fig3.jpg" /> Şekil 3 peroksidatif Fenton footprinting reaksiyon sonra RNA parçaları Analizi. (A) RNA hidroksil radikalleri maruz kalmış ve parçalanma ürünleri denatüre poliakrilamid jel elektroforezi (PAGE) ile çözüldü. Resim, Mg (II) artan konsantrasyonu ile peroksidatif parçalanma ürünleri gösterir. Referans ve kontrol şeritleri etiketli. Bu görüntü dosyası, her bir band hacmi quantitates SAFA programı için girdi. (B) &#39;termal&#39; komplo SAFA tarafından oluşturulur. (C) SAFA nükleotid sayısı çıkış entegre bant yoğunluğu ile ilgili bir tablo değerleri Hill equati olarak bağlayıcı bir model ile analiz(1). Koruma Bölgeleri, Mg (II) konsantrasyonunun bir fonksiyonu olarak bant ayrılmaz yoğunluğunda artışa göre seçilmiştir. K d RNA yarısı izlenmekte olan site katlanmış olduğu Mg (II) konsantrasyonu. Hill katsayısı, n H, bağlayıcı cooperativity hakkında bilgi verir ve bu özel site katlayarak yer alan magnezyum iyonlarının sayısı için daha düşük bir tahmin sağlar eğrisinin eğimi bir ölçüsüdür. <img alt="Tablo 1" src="/files/ftp_upload/3244/3244table1.jpg" /> Tablo 1. Temsilcisi hacimleri farklı Mg (II) konsantrasyonu içeren RNA örnekleri üretmek için.

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Enzym. 130, 132-181 (1986).</li><li>Shcherbakova, I., Mitra, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydroxyl-radical+footprinting+to+probe+equilibrium+changes+in+RNA+tertiary+structure.">Hydroxyl-radical footprinting to probe equilibrium changes in RNA tertiary structure.</a> Methods. Enzym. 468, 31-46 (2009).</li><li>Howlett, G. J., Minton, A. P., Rivas, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analytical+ultracentrifugation+for+the+study+of+protein+association+and+assembly.">Analytical ultracentrifugation for the study of protein association and assembly.</a> Curr. Opinion. Chem. Biol. 10, 430-436 (2006).</li><li>McGinnis, J. L., Duncan, C. D., Weeks, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+SHAPE+and+hydroxyl+radical+analysis+of+RNA+structure+and+ribonucleoprotein+assembly.">High-throughput SHAPE and hydroxyl radical analysis of RNA structure and ribonucleoprotein assembly.</a> Methods. Enzym. 468, 67-89 (2009).</li><li>Jonikas, M. A., Radmer, R. J., Laederach, A., Das, R., Pearlman, S., Herschlag, D., Altman, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coarse-grained+modeling+of+large+RNA+molecules+with+knowledge-based+potentials+and+structural+filters.">Coarse-grained modeling of large RNA molecules with knowledge-based potentials and structural filters.</a> RNA. 15, 189-199 (2009).</li></ol>

Çıkar çatışması ilan etti.

Hidroksil radikal footprinting nükleik asitlerin çözücü erişilebilir yüzey alanı değerlendirmek için değerli bir araçtır. Üçüncül yapısı 14 Kalitatif ve kantitatif oluşumu, iyon tipi ve konsantrasyonu, pH, sıcaklık, bağlayıcı proteinler veya katlama ko-faktörler gibi parametrelerin bir fonksiyonu olarak takip edilebilir . Yalındır ve ucuz bir protokol ve çıkan solvent erişilebilirlik ve tek nükleotid düzeyde katlanır bilgi zorlayıcı bir kombinasyon bu yöntem çok cazip hal...

<table border="1">
	<tbody>
		<tr>
			<th>Name</th>
			<th>Company</th>
			<th>Cat#</th>
		</tr>
		<tr>
			<td>Sodium Cacodylate
			(Caution! Toxic)	 	
			</td>
			<td>Sigma</td>
			<td>C4945-25g</td>
		</tr>
		<tr>
			<td>EDTA (0.5 M)</td>
			<td>Ambion</td>
			<td>AM9260G</td>
		</tr>
		<tr>
			<td>DEPC treated water</td>
			<td>Ambion</td>
			<td>AM9915G</td>
		</tr>
		<tr>
			<td>Sodium Acetate (3 M)</td>
			<td>Ambion</td>
			<td>AM9740</td>
		</tr>
		<tr>
			<td>MgCl2 (1 M)</td>
			<td>Ambion</td>
			<td>AM9530G</td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>Ambion</td>
			<td>AM9902</td>
		</tr>
		<tr>
			<td>Sodium Citrate</td>
			<td>Sigma</td>
			<td>W302600</td>
		</tr>
		<tr>
			<td>tRNA</td>
			<td>Sigma</td>
			<td>R-7876</td>
		</tr>
		<tr>
			<td>Sodium-L-ascorbate</td>
			<td>Sigma</td>
			<td>A7631-25g</td>
		</tr>
		<tr>
			<td>Fe(NH4)2(SO4)2 . 6 H2O</td>
			<td>Sigma</td>
			<td>F1543-500g</td>
		</tr>
		<tr>
			<td>RNase T1</td>
			<td>Fermentas</td>
			<td>EN0541</td>
		</tr>
		<tr>
			<td>Hydrogen Peroxide (30%)</td>
			<td>Sigma</td>
			<td>349887</td>
		</tr>
	</tbody>
</table>

monitoring equilibrium changes in rna structure by 'peroxidative' and 'oxidative' hydroxyl radical footprinting

RNA molekülleri, biyolojide önemli bir rol oynamaktadır. , RNA genetik bilginin iletilmesi için ek olarak, regülatör, bağlayıcı veya katalizör olarak belirli bir biyolojik rolünü yerine getiren benzersiz üçüncül yapıları içine katlayabilirsiniz. Üçüncül temas oluşumu hakkında bilgi RNA molekülleri işlevini anlamak için önemlidir. Hidroksil radikalleri (• OH), yüksek reaktivite ve küçük boyutları nedeniyle nükleik asitlerin yapısını benzersiz probları 1 footprinting prob olarak kullanıldığında, hidroksil radikalleri DNA 1 ve RNA&#39;nın 2 fosfodiester omurgası solvent erişilebilir yüzey haritası tek nükleotid çözünürlük gibi ince. Hidroksil radikal footprinting moleküller arası bir temas yüzeyi içerisinde nükleotidler, DNA-protein 1 ve RNA-protein komplekslerinin örneğin tanımlamak için kullanılır. Denge 3 ve 4 geçişler kinetik bir soluti bir fonksiyonu olarak hidroksil radikal footprinting yaparak tespit edilebilirdeğişken veya sırasıyla. Footprinting bir anahtar özelliği probu bu sınırlı maruz kalma (örneğin, &#39;tek-hit kinetiği), polimerin her bir nükleotid üniforması örnekleme sonuçları 5 Bu video makalede, RNA numune hazırlama ve Mg (II)-aracılı bir katlama izotermleri belirlenmesi göstermek için Tetrahymena ribozim P4-P6 etki alanı kullanın . H 2 O 2 (bu &#39;peroksidatif&#39; protokolünün diyoruz) ve doğal olarak çözünmüş O 2 kullanan bir değerli, ancak yaygın olarak bilinen, alternatif (biz bu &#39;gerektiren bilinen bir hidroksil radikal footprinting protokolü kullanımını tanımlamak oksidatif &#39;protokolü). Veri azaltma, dönüşüm ve analiz işlemleri genel bir bakış sunulmuştur.

Watch this Scientific Journal Video about Monitoring Equilibrium Changes in RNA Structure by 'Peroxidative' and 'Oxidative' Hydroxyl Radical Footprinting at JoVE.com

Monitoring Equilibrium Changes in RNA Structure by &#039;Peroxidative&#039; and &#039;Oxidative&#039; Hydroxyl Radical Footprinting

Bu protokol, Mg (II) bağlı hidroksil radikal footprinting iki yöntem RNA üçüncül yapısının oluşumu ölçmek için nasıl kullanılacağını açıklar.

RNA Yapısı &#39;peroksidatif&#39; ve &#39;Oksidatif&#39; Hidroksil Radikal footprinting İzleme Denge Değişiklikler

RNA molecules play an essential role in biology. In addition to transmitting genetic information, RNA can fold into unique tertiary structures fulfilling ...

monitoring-equilibrium-changes-rna-structure-peroxidative-oxidative

Research

JoVE Journal

Biology

Monitoring Equilibrium Changes in RNA Structure by 'Peroxidative' and 'Oxidative' Hydroxyl Radical Footprinting

14.2K Görüntüleme.  Hunter College. Bu protokol, Mg (II) bağlı hidroksil radikal footprinting iki yöntem RNA üçüncül yapısının oluşumu ölçmek için nasıl kullanılacağını açıklar.

Video: Monitoring Equilibrium Changes in RNA Structure by 'Peroxidative' and 'Oxidative' Hydroxyl Radical Footprinting

Single Molecule Methods for Monitoring Changes in Bilayer Elastic Properties

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein interactions and more general lipid bilayer-protein interactions. These interactions are particularly important in pharmacological research, as many current pharmaceuticals on the market can alter the lipid bilayer material properties, which can lead to altered membrane protein function.   The formation of gramicidin channels are dependent on conformational changes in gramicidin subunits which are in turn dependent on the properties of the lipid.  Hence the gramicidin channel  current is a reporter of altered properties of the bilayer due to certain compounds.  

Membrane protein function is regulated by the cell membrane lipid composition. This video-article details how to form a patch using bilayer patch electrodes, as well as how to use gramicidin channels as reporters of altered membrane properties.

Çift katlı Elastik Özellikleri İzleme Değişiklikler için Tek Molekül Yöntemleri

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein ...

Biyoloji

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 &mu;M4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.

Apoptozis sırasında Mitokondrial Kalsiyum Düzeyleri Dinamik değişiklikler izlenmesi Genetiği Kodlanmış Kalsiyum Sensor Kullanımı

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

HeLa Hücre Transfeksiyon Ardından Gaussia Lusiferaz mRNA in vitro transkripsiyon ve Kapatma yılında

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

Kabuklu uzun vadeli Lethal Toksisite Testi Artemia Franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

RNA Saflaştırma tarafından Kromatin İzolasyon (chirp)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

Floresans tarafından viral RNA tespit edilmesi<em&gt; In situ</em&gt; Hibridizasyon (FISH)

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

RNA Secondary Structure Prediction Using High-throughput SHAPE

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed "high-throughput selective 2' hydroxyl acylation analyzed by primer extension", or SHAPE, allows prediction of RNA secondary structure with single nucleotide resolution. This approach utilizes chemical probing agents that preferentially acylate single stranded or flexible regions of RNA in aqueous solution. Sites of chemical modification are detected by reverse transcription of the modified RNA, and the products of this reaction are fractionated by automated capillary electrophoresis (CE). Since reverse transcriptase pauses at those RNA nucleotides modified by the SHAPE reagents, the resulting cDNA library indirectly maps those ribonucleotides that are single stranded in the context of the folded RNA. Using ShapeFinder software, the electropherograms produced by automated CE are processed and converted into nucleotide reactivity tables that are themselves converted into pseudo-energy constraints used in the RNAStructure (v5.3) prediction algorithm. The two-dimensional RNA structures obtained by combining SHAPE probing with in silico RNA secondary structure prediction have been found to be far more accurate than structures obtained using either method alone.

High-throughput selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) utilizes a novel chemical probing technology, reverse transcription, capillary electrophoresis and secondary structure prediction software to determine the structures of RNAs from several hundred to several thousand nucleotides at single nucleotide resolution.

Yüksek verim SHAPE kullanarak RNA İkincil Yapısı Tahmin

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

Saf Yabanitip ve Mutant CFTR Protein Fonksiyonel Sulandırma ve Kanal Aktivite Ölçümleri

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Tracking Drug-induced Changes in Receptor Post-internalization Trafficking by Colocalizational Analysis

The intracellular trafficking of receptors is a collection of complex and highly controlled processes. Receptor trafficking modulates signaling and overall cell responsiveness to ligands and is, itself, influenced by intra- and extracellular conditions, including ligand-induced signaling. Optimized for use with monolayer-plated cultured cells, but extendable to free-floating tissue slices, this protocol uses immunolabelling and colocalizational analysis to track changes in intracellular receptor trafficking following both chronic/prolonged and acute interventions, including exogenous drug treatment. After drug treatment, cells are double-immunolabelled for the receptor and for markers for the intracellular compartments of interest. Sequential confocal microscopy is then used to capture two-channel photomicrographs of individual cells, which are subjected to computerized colocalizational analysis to yield quantitative colocalization scores. These scores are normalized to permit pooling of independent replicates prior to statistical analysis. Representative photomicrographs may also be processed to generate illustrative figures. Here, we describe a powerful and flexible technique for quantitatively assessing induced receptor trafficking.

Receptor trafficking modulates signaling and cell responsiveness to ligands and is, itself, responsive to cell conditions, including ligand-induced signaling. Here, we describe a powerful and flexible technique for quantitatively assessing drug-induced receptor trafficking using immunolabeling and colocalizational analysis.

Colocalizational Analizi Reseptör Sonrası içselleştirme Ticareti Uyuşturucu kaynaklı Değişiklikleri İzleme

The intracellular trafficking of receptors is a collection of complex and highly controlled processes. Receptor trafficking modulates signaling and ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Fekal Glukokortikoid Analizi: Tek tırnaklılarda in Non-invaziv Adrenal İzleme

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...