S. Lusvarghi, J. Sztuba-Solinska, KJ Purzycka, JW Rausch ve SFJ Le Grice Ulusal Kanser Enstitüsü, Ulusal Sağlık Enstitüleri, ABD İntramural Araştırma Programı tarafından desteklenmektedir.

<ol>
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Çıkar çatışması ilan etti.

Burada yüksek verimli SHAPE için ayrıntılı bir protokol, her boyutta RNA&#39;lar için tek nükleotid çözünürlük sekonder yapı tayini sağlayan bir teknik size sunmak. Ayrıca, ikincil yapı tahmini algoritmaları ile deneysel SHAPE veri bağlantısı tek başına ya yöntemiyle mümkün olandan daha yüksek doğruluk derecesi ile RNA 2D modellerin üretimi kolaylaştırır. Floresan-etiketli primerlerin ve otomatik CE kombinasyonu tek bir deneyde uzun RNA sekanslarının, çözünürlük kolaylaştırmak gel...

Yapıştırma, çeviri, virüs çoğaltma ve kanser düzenlenmesinde yer alan katalitik olmayan kodlama RNA&#39;lar işlevlerini anlamak için, RNA yapısının detaylı bilgi 1,2 gereklidir. Ne yazık ki, RNA katlama doğru tahmin müthiş bir meydan okuma sunuyor. Klasik tarama maddeler muzdarip toksisite, eksik nükleotid kapsamı ve / veya deney başına 100-150 nükleotidleri ile sınırlı verim gibi birçok dezavantajları. Çıplak ikincil yapı tahmini algoritmaları benzer, dezavantajlı etkili enerjik benzer yapıları arasında ayırt etmek için kendi yetersizlik kaynaklanan yanlışlıklar nedeniyle vardır. Özellikle büyük RNA&#39;lar, aynı zamanda X-ışını kristalografisi ve şekilsel esnekliği ve bu teknikler için gerekli son derece saf bir numune büyük miktarlarda nedeniyle spektroskopisi (NMR) nükleer manyetik rezonans olarak 3D yapı belirleme yöntemleri genellikle alınamamaktadır. High verimli SHAPE tek nükleotid çözünürlükte büyük RNA&#39;lar yapıları sondalama için etkili, basit bir yaklaşım sağlayarak bu sorunların çoğu çözer. Ayrıca, şekil için kullanılan reaktiflere işlemek için güvenli, kolay ve reaktifler tarama çoğu diğer kimyasal aksine, dört ribonükleotidler ile reaksiyona girerler. Bu reaktifler de mümkün bağlamda in vivo (lar) 3 kendi RNA&#39;lar araştırmak için yapım, hücre membranları içine nüfuz edebilir. Başlangıçta hafta laboratuar 4 geliştirilen ŞEKLİ RNA&#39;lar, geniş bir yelpazede ~ 9 kb HIV-1 RNA genomunun 5 tam ikincil yapısının belirlenmesi; en önemli örneği analiz etmek için kullanılmıştır. Şeklini kullanarak Diğer başarıları bulaşıcı viroidler 6, insan uzun kodlayıcı olmayan RNA&#39;lar 7, maya ribozomlar 8 ve 9 riboswitches hem de tespit etmek virionu ile ilişkili HIV-1 RNA 3 protein bağlanma yerleri yapılarının aydınlatılması içerir. WhSHAPE protokol Ile orijinal ve yüksek verimli varyasyonlar başka bir yerde 10-12 yayınlanmıştır, Bu çalışmada floresan oligonükleotidler, Beckman Coulter CEQ 8000 Genetic Analyzer kullanarak yüksek verimli SHAPE RNA ikincil yapı tayini ayrıntılı bir açıklamasını sağlar ve SHAPEfinder ve RNAStructure (v5.3) yazılımı. Daha önce yayınlanmamış teknik ayrıntı ve sorun giderme tavsiye de dahildir. SHAPE varyasyonları ŞEKİL ve varyasyonları özü seçici modifikasyonu sitelerinin hantal katılma ürünleri üreten, 2&#39;-hidroksi (2&#39;-OH) riboz grupları açilleme elektrofilik anhidritler, sulu çözelti içinde RNA maruz kalmasıdır. Baz eşleştirilmiş veya mimari Yapım ise bu kimyasal reaksiyon, tek iplikli nükleotidler bu reaktif ile elektrofilik saldırı için elverişli konformasyonlar benimsemeye daha yatkındır gibi, yerel RNA yapısal dinamikleri sorgulayan bir araç olarak hizmet vermektedirained nükleotid daha az ya da 10 reaktif değillerdir. Adukt formasyonu Yer modifiye RNA (&quot;(+)&quot; primer uzatma reaksiyonu) ile ilgili belirli bir site hibridize floresan veya radyoaktif primerlerle başlatarak, ters transkripsiyon ile tespit edilir. Ters transkriptaz (RT) asile ribonucleotides hareket başarısız olduğunda, cDNA ürünleri bir havuz olan uzunlukları değişiklik siteleri denk üretilir. Bir kontrol, &quot;(-)&quot; astar uzantısı reaktif maruz olmamıştır RNA kullanarak reaksiyon da çok yapılır bu yapı, non-spesifik RNA zincir kırıkları, vb olabilir RNA nedeniyle DNA sentezi (yani &quot;durur&quot;) erken sona ermesi. kimyasal modifikasyonu ile üretilen duraklatma ayırt edilebilir. Son olarak, aynı primerlerle başlatılması iki-dideoksi sekanslama reaksiyonları RNA primer dizisi elektroforezi ile aşağıdaki reaktif nükleotid ilişkilendirmek için marker olarak kullanılmaktadır. SHAP orijinal uygulamasında(-) Ve iki dizi reaksiyonlar E aynı 32 P-uç-etiketli astar, (+) için kullanılmaktadır. Bu reaksiyonların ürünleri bir% 5-8 poliakrilamid jel levha bitişik kuyu içine yüklenir ve poliakrilamid jel elektroforezi (PAGE, Şekil 1), denatüre edici ile fraksiyone edilmiştir. Geleneksel şekli ile üretilen jel görüntüleri kantitatif analiz SAFA, yarı-otomatik bir ayak izi analiz yazılımı 13 kullanılarak da gerçekleştirilebilir. Buna karşılık, yüksek verimli SHAPE floresan etiketli astar ve otomatik kapiller elektroforez kullanır. Spesifik olarak, inceleme altındaki RNA ortak bir dizi ancak farklı 5 &#39;floresan etiketler olarak sentezlenebilir ya da satın alınması gerekir olan dört DNA primerleri kümesi her bir bölgesi için. Bu farklı-etiketli oligonükleotidler asal iki SHAPE reaksiyonlar ve iki sıralama reaksiyonları, toplanmış ve fraksiyone / otomatik kapiller elektroforez (CE) tarafından tespiti yapılan ürünler için hizmet vermektedir. NereyeEAS RNA 100-150 nt reaktivite profili temel yaklaşımı kullanarak, dört reaksiyonlar bir dizi elde edilebilir, yüksek verimli bir şekli, bir tek numune toplanmış 3 300-600 nt çözünürlüğü sağlar. Gibi pek çok 96 gibi, örnekler 12 ardışık CE çalışır (Şekil 2) boyunca fraksiyonasyon için hazırlanmış olabilir iken, aynı zamanda, fraksiyonlara ayrılmış olabilir üste reaksiyonlar 8 setleri için. Ayrıca, CEQ ve diğer genetik analiz çıkan verilerin işlenmesi ve analiz etmek için geliştirilen SHAPEfinder yazılım, daha otomatik ve SAFA 13 veya diğer jel-analiz paketleri çok daha az kullanıcı müdahalesi gerektirir. Daha gelişmiş yüksek verimli yöntemleri son zamanlarda elde etmek için teknikler sıralama yeni nesil ile birlikte yapı-belirli enzimler yerine alkilleme reaktifleri kullanmak PARS (RNA yapısı paralel analiz) 14 ve Frag-Seq (fragman-dizi) 15, olarak ortaya çıkmıştır BİLİŞİMRNA yapısı hakkında n. Bu tekniklerin çekiciliğini rağmen, sondalama nükleaz doğasında birçok sınırlamalar hala 16 kalır. Bu sorunlar, yeni nesil sıralama geleneksel SHAPE&#39;e gerçekleştirilen edilene benzer bir şekilde kimyasal modifikasyonu ve RNA&#39;ların ters transkripsiyon öncesinde ŞEKLİ dizi analizleri (Şekil-Seq) 17 protokol bölgesi atlatılabilir. Bu yöntemler RNA yapı tayini geleceği temsil olsa da, bu yeni nesil sıralama çok pahalı ve birçok laboratuvarlara kullanılamaz durumda olduğunu hatırlamak önemlidir. SHAPE Veri Analizi Genetik analiz üretilen veri taşıma zaman indeksi karşı çizilen kılcal detektörü akan numunenin floresan yoğunluğu (ler) in bir electropherogram şeklinde sunulmuştur. Bu grafik, dört flüoresan kanala tekabül eden üst üste izleri formunda alırin farklı bir florofor tespit etmek için kullanılır ve tek tek her bir iz cDNA&#39;sı ya da sekanslama ürünlerine karşılık gelen tepe oluşur burada. Elektroferogram veri bir sekme ile sınırlandırılmış metin dosyası olarak genetik analiz ihraç ve ShapeFinder dönüşüm ve analiz yazılımı 18 alınır. ShapeFinder ilk geçiş süreleri ve yüksek miktarlar doğru sırasıyla reaksiyon ürünleri, kimlikleri ve miktarları yansıtmasını sağlamak için veri matematiksel dönüşümleri bir dizi gerçekleştirmek için kullanılır. Peaks sonra uyumlu ve entegre ve sonuçlar birincil RNA dizisi ile birlikte tablo vardır. RNA ilgili bölümü için bir &quot;reaktivite profili&quot; (+) nükleotid Her RNA ile ilişkili değerleri kontrol değerleri çıkarılarak, ve aşağıda tarif edildiği gibi verileri normalize edilmesiyle elde edilir. Bu profil normalize tepkime val dönüştürür (v5.3) yazılım 19,20, RNAstructure alınırRNA ikincil yapı katlama algoritması dahil edilmiştir sözde enerji kısıtlamaları içine ues. Bu şekilde algoritma sondalama ve katlama kimyasal birleştiren önemli ölçüde tek başına 12,21 ya yöntemine göre yapı tahmini doğruluğunu artırır. RNAstructure çıktısı (v5.3) en düşük enerji görüntüleri içeren RNA ikincil yapılar metin nokta-dirsek gösterimde SHAPE reaktivite profil (ler), hem de aynı yapılarla renk kodlu. İkincisi daha sonra bu Varna 22 ve PseudoViewer 23 olarak RNA&#39;nın ikincil yapının grafik ekran için özel yazılım ihraç edilebilir. <img alt="Şekil 1" fo:content-width="4.8in" fo:src="/files/ftp_upload/50243/50243fig1highres.jpg" src="/files/ftp_upload/50243/50243fig1.jpg" /> Şekil 1. SHAPE 4,10 ile RNA yapısı belirlenmesi Akış Şeması. (A) RNA mAy biyolojik numunelerin veya in vitro transkripsiyonu sonucunda elde edilebilir. (B) kaynağına bağlı olarak, RNA, katlanmış ya da başka şekilde işlem ve ŞEKİL reaktifi ile modifiye edilir. (C) ters transkripsiyon floresan veya radyoaktif olarak etiketlenmiş primerler kullanılarak. (D) cDNA ürünlerdir ya kılcal veya levha jel tabanlı elektroforezi ile fraksiyonlara ayrılmış. (E) Parça analizi. (F) RNA yapısı tahmini. <a href="http://www.jove.com/files/ftp_upload/50243/50243fig1large.jpg" target="_blank">büyük rakam görmek için buraya tıklayın.</a> <img alt="Şekil 2," fo:content-width="6in" fo:src="/files/ftp_upload/50243/50243fig2highres.jpg" src="/files/ftp_upload/50243/50243fig2.jpg" /> Şekil 2. CE tabanlı SHAPE yüksek verimli karakter hızlı çoklu RNA&#39;lar analizi ve / veya aynı RNA oluşan parçalara ayır sağlar. (A) </stRong&gt; bir RNA 300-600 nt bölümler (renk yeşil, mavi ve kırmızı kodlu) (B) RNA bölümleri floresan astar farklı ayarlar (siyah ok) ile bağımsız tanınacak olan (C) takımları ayrılabilir nasıl temsil reaksiyonları ~ 3 kb RNA1 için tam koruma sağlar, sırasıyla kuyu A1, B1, C1, vb, içine toplanmış ve yüklenir. RNA&#39;lar 2, 3, 4, vb reaksiyon ürünleri benzer ardışık elektroforetik çalıştırır fraksiyon için hazırlanabilir. <a href="http://www.jove.com/files/ftp_upload/50243/50243fig2large.jpg" target="_blank">büyük rakam görmek için buraya tıklayın.</a>

<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>N-methylisatoic anhydride (NMIA)</td>
			<td>Life technologies</td>
			<td>M25</td>
			<td>Dissolve in anhydrous DMSO</td>
		</tr>
		<tr>
			<td>1-methyl-t-nitroisatoic anhydride (1M7)</td>
			<td>see ref. 22</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Superscript III Reverse Transcriptase</td>
			<td>Life technologies</td>
			<td>18080044</td>
			<td>10,000 units</td>
		</tr>
		<tr>
			<td>Thermo sequenase cycle sequencing kit</td>
			<td>Affymetrix</td>
			<td>78500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Materials provided by the user</td>
		</tr>
		<tr>
			<td>RNA of interest</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>6 pmol per reaction (the limit of detection will be determined by the instrument)</td>
		</tr>
		<tr>
			<td>Sets of four 5' labeled primers (Cy5, Cy5.5, WellRed D2 and WellRed D1/Licor IR800)</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Primers are complementary to the RNA and are used in reverse transcription and sequencing reactions. The listed fluorophores are optimal for the Beckman Coulter 8000 CEQ. Primers may be purchased or synthesized in house.</td>
		</tr>
		<tr>
			<td>DNA template</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>DNA is used for sequencing reactions, and must contain the sequence of the RNA being studied - including any 3'terminal extension, if present. Where applicable, it is often convenient to use the RNA transcription template.</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Buffers</td>
		</tr>
		<tr>
			<td>10x RNA renaturation buffer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>100 mM Tris-HCl pH 8.0, 1 M KCl, 1 mM EDTA</td>
		</tr>
		<tr>
			<td>5X RNA folding buffer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>200 mM Tris-HCl pH 8.0, 25 mM MgCl2, 2.5 mM EDTA, 650 mM KCl. (This buffer might be changed depending on the case (e.g. pH, EDTA, Mg, RNase inhibitor)</td>
		</tr>
		<tr>
			<td>2.5X RT mix</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>4 &mu;l 5X buffer, 1 &mu;l 100 mM DTT, 1.5 &mu;l water,1 &mu;l 10 mM dNTPs, 0.5 &mu;l SuperScript III. Note that the 5X buffer and 100 mM DTT are provided with purchase of SuperScript III (Invitrogen).</td>
		</tr>
		<tr>
			<td>GenomeLab Sample Loading Solution (Beckman Coulter)</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Attention: Avoid multiple freeze-thaw cycles</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>Capillary electrophoresis</td>
			<td>Beckman</td>
			<td>CEQ8000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Thermocycler</td>
			<td>varies</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

rna secondary structure prediction using high-throughput shape

Biyolojik süreçlerinde RNA işlevini anlama RNA yapısı ayrıntılı bir bilgi gerektirir. Bu sonuna doğru, dublaj metodoloji, veya SHAPE &quot;astar uzantısı ile analiz yüksek verimli seçici 2 &#39;hidroksil asilleme&quot;, tek nükleotid çözünürlükte RNA ikincil yapı tahmini sağlar. Bu yaklaşım, tercihen sulu çözelti içinde RNA tek sarmallı ya da esnek bölgeler asil ile kimyasal tarama maddeler kullanır. Bir kimyasal modifikasyon Yer modifiye RNA ters transkripsiyon ile tespit edilir ve bu reaksiyonun ürünleri, otomatik kapiler elektroforez (CE) ile fraksiyone edilmiştir. Ters transkriptaz ŞEKİL reaktifleri ile değiştirilmiş olanlar RNA nükleotid duraklar yana Sonuçta elde edilen cDNA kütüphanesi dolaylı olarak tek kapandı RNA bağlamında mahsur bu ribonükleotidler eşler. ShapeFinder yazılımı kullanarak, otomatik CE tarafından üretilen electropherograms işlenmiş ve nu dönüştürülürcleotide tepkime tablolar kendilerini RNAStructure (v5.3) tahmini algoritması kullanılan sözde enerji kısıtlamaları dönüştürülür. Siliko RNA ikincil yapı tahmininde ile sondalama SHAPE birleştirerek elde edilen iki boyutlu RNA yapıları tek başına her iki yöntemi kullanarak elde edilen yapılara göre çok daha doğru olduğu tespit edilmiştir.

RNA mevcudiyetinde, HIV-1 devir tepki elemanı (RRE) ve bir 3 &#39;terminal yapısı kaseti 4 bunun 37&#39; de ısıtma, soğutma ve inkübasyon tarafından katlanmış ve bundan sonra in vitro transkripsiyon ve doğrusallaştırılmış bir plasmidden elde edilmiştir ° C ihtiva eden MgCl2. RNA NMIA maruz bırakıldı ve daha sonra 3 &#39;terminal yapısı kaseti hibridize bir 5&#39;-uç-etiketli DNA primeri transkripsiyonu ters. Sonuçta ortaya çıkan şekil cDNA kütüphanesi...

Watch this Scientific Journal Video about RNA Secondary Structure Prediction Using High-throughput SHAPE at JoVE.com

RNA Secondary Structure Prediction Using High-throughput SHAPE

Astar uzantısı (SHAPE) tarafından analiz yüksek verimli seçici 2 &#39;hidroksil asilleme tek nükleotid çözünürlükte binlerce nükleotid birkaç yüz ile RNA&#39;lar yapılarını belirlemek için bir teknoloji sondalama yeni kimyasal, ters transkripsiyon, kapiler elektroforez ve ikincil yapı tahmini yazılımı kullanır.

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 ...

rna-secondary-structure-prediction-using-high-throughput-shape

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Transcription: DNA to RNA

SCIENTISTS IN ACTION

31.2K Görüntüleme.  Frederick National Laboratory for Cancer Research. Astar uzantısı (SHAPE) tarafından analiz yüksek verimli seçici 2 'hidroksil asilleme tek nükleotid çözünürlükte binlerce nükleotid birkaç yüz ile RNA'lar yapılarını belirlemek için bir teknoloji sondalama yeni kimyasal, ters transkripsiyon, kapiler elektroforez ve ikincil yapı tahmini yazılımı kullanır.

Video: RNA Secondary Structure Prediction Using High-throughput SHAPE

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

Yüksek verim Maya Plazmid overekspresyonu Ekran

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

Biyoloji

A Protocol for Computer-Based Protein Structure and Function Prediction

Genome sequencing projects have ciphered millions of protein sequence, which require knowledge of their structure and function to improve the understanding of their biological role. Although experimental methods can provide detailed information for a small fraction of these proteins, computational modeling is needed for the majority of protein molecules which are experimentally uncharacterized. The I-TASSER server is an on-line workbench for high-resolution modeling of protein structure and function. Given a protein sequence, a typical output from the I-TASSER server includes secondary structure prediction, predicted solvent accessibility of each residue, homologous template proteins detected by threading and structure alignments, up to five full-length tertiary structural models, and structure-based functional annotations for enzyme classification, Gene Ontology terms and protein-ligand binding sites. All the predictions are tagged with a confidence score which tells how accurate the predictions are without knowing the experimental data. To facilitate the special requests of end users, the server provides channels to accept user-specified inter-residue distance and contact maps to interactively change the I-TASSER modeling; it also allows users to specify any proteins as template, or to exclude any template proteins during the structure assembly simulations. The structural information could be collected by the users based on experimental evidences or biological insights with the purpose of improving the quality of I-TASSER predictions. The server was evaluated as the best programs for protein structure and function predictions in the recent community-wide CASP experiments. There are currently &gt;20,000 registered scientists from over 100 countries who are using the on-line I-TASSER server.

Guidelines for computer based structural and functional characterization of protein using the I-TASSER pipeline is described. Starting from query protein sequence, 3D models are generated using multiple threading alignments and iterative structural assembly simulations. Functional inferences are thereafter drawn based on matches to proteins with known structure and functions.

Bilgisayar Tabanlı Protein Yapısı ve İşlevi Tahmin Protokolü

Genome sequencing projects have ciphered millions of protein sequence, which require knowledge of their structure and function to improve the ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

Yüksek verim Kristalizasyon lipidik Bicelle Yöntemiyle Membran Proteinleri

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place over the next 5 years. For example, the sequencing of the genomes for 15 malaria mosquitospecies is currently being done using an Illumina platform3,4. This Anopheles species cluster includes both vectors and non-vectors of malaria. When the genome assemblies become available, researchers will have the unique opportunity to perform comparative analysis for inferring evolutionary changes relevant to vector ability. However, it has proven difficult to use next-generation sequencing reads to generate high-quality de novo genome assemblies5. Moreover, the existing genome assemblies for Anopheles gambiae, although obtained using the Sanger method, are gapped or fragmented4,6.
Success of comparative genomic analyses will be limited if researchers deal with numerous sequencing contigs, rather than with chromosome-based genome assemblies. Fragmented, unmapped sequences create problems for genomic analyses because: (i) unidentified gaps cause incorrect or incomplete annotation of genomic sequences; (ii) unmapped sequences lead to confusion between paralogous genes and genes from different haplotypes; and (iii) the lack of chromosome assignment and orientation of the sequencing contigs does not allow for reconstructing rearrangement phylogeny and studying chromosome evolution. Developing high-resolution physical maps for species with newly sequenced genomes is a timely and cost-effective investment that will facilitate genome annotation, evolutionary analysis, and re-sequencing of individual genomes from natural populations7,8.
Here, we present innovative approaches to chromosome preparation, fluorescent in situ hybridization (FISH), and imaging that facilitate rapid development of physical maps. Using An. gambiae as an example, we demonstrate that the development of physical chromosome maps can potentially improve genome assemblies and, thus, the quality of genomic analyses. First, we use a high-pressure method to prepare polytene chromosome spreads. This method, originally developed for Drosophila9, allows the user to visualize more details on chromosomes than the regular squashing technique10. Second, a fully automated, front-end system for FISH is used for high-throughput physical genome mapping. The automated slide staining system runs multiple assays simultaneously and dramatically reduces hands-on time11. Third, an automatic fluorescent imaging system, which includes a motorized slide stage, automatically scans and photographs labeled chromosomes after FISH12. This system is especially useful for identifying and visualizing multiple chromosomal plates on the same slide. In addition, the scanning process captures a more uniform FISH result. Overall, the automated high-throughput physical mapping protocol is more efficient than a standard manual protocol.

High-throughput Physical Mapping of Chromosomes using Automated in situ Hybridization

Genome assemblies based on massively parallel DNA sequencing technologies are usually highly fragmented. The development of physical chromosome maps can potentially improve genome assemblies. Here, we demonstrate innovative approaches to chromosome preparation, fluorescent in situ hybridization, and imaging that significantly increase throughput of the physical map development.

Otomatik kullanarak Kromozomlar Yüksek throughput Fiziksel Haritalama<em&gt; In situ</em&gt; Hibridizasyon

Projects to obtain whole-genome sequences for 10,000 vertebrate species1 and for 5,000 insect and related arthropod species2 are expected to take place ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

Bir ev yapımı Çift kızdırma Lusıferaz Testi kullanarak yüksek verim Fonksiyonel Taraması

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.

The zebrafish is an excellent experimental organism to study vertebrate developmental processes and model human disease. Here, we describe a protocol on how to perform a manual high-throughput chemical screen in zebrafish embryos with a whole-mount in situ hybridization (WISH) read-out.

Zebra balığı Embriyolar kullanılarak Yüksek verim Yaklaşıyor A Manuel Küçük Molekül Ekran

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology ...

Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide resolution. This technique captures miRNAs by attaching 3&#8217; and 5&#8217; oligonucleotide adapters to miRNA molecules and allows de novo miRNA discovery. Coupling with powerful next-generation sequencing platforms, miR-Seq has been instrumental in the study of miRNA biology. However, significant biases introduced by oligonucleotide ligation steps have prevented miR-Seq from being employed as an accurate quantitation tool. Previous studies demonstrate that biases in current miR-Seq methods often lead to inaccurate miRNA quantification with errors up to 1,000-fold for some miRNAs1,2. To resolve these biases imparted by RNA ligation, we have developed a small RNA ligation method that results in ligation efficiencies of over 95% for both 3&#8217; and 5&#8242; ligation steps. Benchmarking this improved library construction method using equimolar or differentially mixed synthetic miRNAs, consistently yields reads numbers with less than two-fold deviation from the expected value. Furthermore, this high-efficiency miR-Seq method permits accurate genome-wide miRNA profiling from in vivo total RNA samples2.

MicroRNAs (miRNAs) are a widely conserved class of regulatory molecules. Here we describe a miRNA cloning method that relies upon two potent ligation steps followed by high-throughput sequencing. Our method permits accurate genome-wide quantitation of miRNAs.

Yüksek Verim Dizilimine tarafından MicroRNA ölçümü için Küçük RNA molekülleri, yüksek Verimli ligasyonu

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Post-transkripsiyonel ayarlı genlerin kimyasal Modülatörler için yüksek verimli tarama

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an array of hundreds of queried 3&#8217;UTRs. Target identification is based on the dual-luciferase assay, which detects binding at the mRNA level by measuring translational output, giving a functional readout of miRNA targeting. 3&#8217;LIFE uses non-proprietary buffers and reagents, and publically available reporter libraries, making genome-wide screens feasible and cost-effective. 3&#8217;LIFE can be performed either in a standard lab setting or scaled up using liquid handling robots and other high-throughput instrumentation. We illustrate the approach using a dataset of human 3&#8217;UTRs cloned in 96-well plates, and two test miRNAs, let-7c and miR-10b. We demonstrate how to perform DNA preparation, transfection, cell culture and luciferase assays in 96-well format, and provide tools for data analysis. In conclusion 3&#39;LIFE is highly reproducible, rapid, systematic, and identifies high confidence targets.

Detection of miRNA Targets in High-throughput Using the 3&#039;LIFE Assay

Luminescent identification of functional elements in 3&#8217; untranslated regions (3&#8217;UTRs) (3&#8217;LIFE) is a technique to identify functional regulation in 3&#8217;UTRs by miRNAs or other regulatory factors. This protocol utilizes high-throughput methodology such as 96-well transfection and luciferase assays to screen hundreds of putative interactions for functional repression.

3&#39;LIFE Test kullanma Yüksek throughput miRNA Hedefleri tespiti

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an ...

Identification of Kinase-substrate Pairs Using High Throughput Screening

We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel signal transduction pathways. Our approach features the use of a library of purified GST-tagged human protein kinases and a recombinant protein substrate of interest. We have used this technology to identify MAP/microtubule affinity-regulating kinase 2 (MARK2) as the kinase for a glucose-regulated site on CREB-Regulated Transcriptional Coactivator 2 (CRTC2), a protein required for beta cell proliferation, as well as the Axl family of tyrosine kinases as regulators of cell metastasis by phosphorylation of the adaptor protein ELMO. We describe this technology and discuss how it can help to establish a comprehensive map of how cells respond to environmental stimuli.

Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.

Yüksek Verimli Tarama kullanma Kinaz-substrat Çiftlerinin Kimlik


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...