S. Lusvarghi, J. Sztuba-Solinska, KJ Purzycka, JW Rausch의 및 SFJ 르 Grice는 국립 암 연구소, 건강의 국립 연구소, 미국의 교내 연구 프로그램에 의해 지원됩니다.

<ol>
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관심 없음 충돌 선언하지 않습니다.

우리는 여기서 높은 처리량 SHAPE에 대한 자세한 프로토콜, 어떤 크기의 RNA를위한 단일 염기 해상도로 차 구조 결정을 할 수있는 기법을 제시한다. 또한, 이차 구조 예측 알고리즘을 실험 SHAPE 데이터를 결합하는 것은 혼자 두 방법으로 가능한 것보다 높은 정확도와 RNA 차원 모델의 생성을 촉진한다. 찬란 - 라벨 프라이머 및 자동 CE의 조합은 하나의 실험에서 긴 RNA 시퀀스의 해상도를 촉진 기존의 ?...

접합, 번역, 바이러스 복제 및 암의 조절에 관여 촉매 및 비 코딩 RNA를의 기능을 이해하기 위해, RNA 구조의 상세한 지식은 1,2 필요합니다. 불행하게도, RNA 접이식의 정확한 예측은 강력한 도전을 선물한다. 고전 프로빙 에이전트는 고통과 같은 독성, 불완전 염기 범위 및 / 또는 실험 당 100 ~ 150 뉴클레오티드 제한 처리 등 많은 단점에서. 남의 차 구조 예측 알고리즘은 유사하게 불리한 효율적으로 정력적으로 유사한 구조를 구별하기 위해 무능력으로 인한 부정확성으로 인해 수 있습니다. 특히 큰 RNA를 또한 X-선 결정학과 구조적 유연성과 이러한 기술에 필요한 고순도 샘플을 대량으로 인해 분광법 (NMR) 공명 핵 자기와 같은 3D 구조를 결정하는 방법을 자주 내화물입니다. H고등학교의 처리량 모양의 단일 염기 해상도로 대형의 RNA 구조를 프로빙에 효과적인 간단한 방법을 제공함으로써 많은 문제를 해결합니다. 또한, 모양에 사용되는 시약 처리 안전, 쉽고, 시약 프로브 대부분의 다른 화학 대조적으로, 네 개의 리보 뉴클레오티드로 반응합니다. 이 시약은 또한 가능한 생체 컨텍스트 (들) 3 자신의 RNA를을 조사하기 위해 만들어 세포막을 통과 할 수 있습니다. 원래 주 실험실 4에서 개발, 모양의 RNA 다양한 ~ 9킬로바이트 HIV-1 RNA 게놈 5의 전체 차 구조 결정되는 가장 주목할만한 예제를 분석하는 데 사용되었습니다. SHAPE를 사용하여 다른 주목할만한 업적은 전염성 로이드 6, 인간의 긴 비 코딩 RNAs를 7, 효모 리보솜 8 및 리보 스위치 9뿐만 아니라 식별 할 수 virion의 관련되는 HIV-1 RNA 3 단백질 결합 부위의 구조의 해명이 ​​(가) 있습니다. WHSHAPE 프로토콜의 일 원본과 높은 처리량 변화는 다른 10-12 게시 된, 본 연구는 형광 올리고 뉴클레오타이드를, 베크 만 보습 바로 앞에 달린 풀 베는 CEQ 8000 유전자 분석기를 사용하여 높은 처리량의 형상에 의해 RNA 이차 구조 결정에 대한 자세한 설명을 제공하고, SHAPEfinder 및 RNAStructure (V5.3) 소프트웨어입니다. 이전에 발표되지 않은 기술적 인 세부 사항 및 문제 해결 조언도 포함되어 있습니다. 모양의 변형 SHAPE와 그 변화의 본질은 선택적으로 변형 사이트에서 부피 부가 물을 생산, 2&#39;-히드 록시 (2&#39;-OH) 리보오스 그룹을 acylate 전자 성 무수물로 수용액 RNA의 노출이다. 베이스 쌍 또는 구조적 생성자하는 동안이 화학 반응은 단일 가닥 핵산이 시약에 의한 전자 성 공격에 도움이되는 conformations에 채택하는 경향이기 때문에, 지역의 RNA 구조 역학을 심문하는 수단으로 역할을ained 뉴클레오티드가 작거나 10 반응성이다. 부가 생성물 형성의 사이트는 수정 된 RNA ( &quot;(+)&quot;프라이머 연장 반응)에 특정 사이트에 하이브리드 형광이나 방사성 동위 원소 프라이머에서 시작 역전사에 의해 감지됩니다. 역전사 효소 (RT)는 실화 리보 뉴클레오타이드를 통과하는 데 실패하면, cDNA를 제품의 풀은 그 길이 수정의 사이트와 일치 생산됩니다. 컨트롤은 &quot;(-)&quot;프라이머 확장 시약에 노출되지 않은 RNA를 활용 반응은 그렇게 수행되는 구조 특이 RNA 가닥 파손 등, 5 월을 RNA에 의한 DNA 합성 (예 : &quot;중지&quot;)의 조기 종료. 화학적 변형에 의해 생성 일시 중지 구분할 수. 마지막으로, 같은 프라이머에서 시작 두 dideoxy 시퀀싱 반응은 RNA 기본 순서가 전기를 다음과 같이 반응 뉴클레오티드의 상관 관계를 마커로 사용됩니다. SHAP의 원래 응용 프로그램에서(-), 두 개의 연속 반응 E, 같은 32 P-끝 레이블 프라이머 (+)를 위해 사용됩니다. 이러한 반응의 제품은 5-8 %의 폴리 아크릴 아미드 슬래브 겔에 인접 우물에로드 폴리 아크릴 아마이드 겔 전기 영동 (PAGE, 그림 1) 변성에 의해 분별하고 있습니다. 기존의 모양에 의해 생성 된 겔 이미지의 정량 분석은 사파, 반자동 배출량 측정 분석 소프트웨어 13를 사용하여 수행 할 수 있습니다. 반면, 높은 처리량 모양 찬란 프라이머 및 자동화 모​​세관 전기 영동을 사용합니다. 특히, 조사 대상 RNA, 일반적인 순서 만 다른 5 &#39;형광 라벨을 합성하거나 구입해야하는 데 네 가지 DNA 프라이머 세트의 각 지역. 이러한 다르게 표지 된 올리고 뉴클레오티드는 주요 두 가지 모양의 반응과 두 개의 연속 반응, 풀링 그리고 분별 / 자동 모세관 전기 영동 (CE)에 의해 감지되는의 제품을 제공합니다. 실어EAS는 RNA의 100 ~ 150 NT의 반응 프로파일은 원래의 방법을 사용하여 네 가지 반응의 집합에서 얻을 수있는, 높은 처리량 SHAPE는 단일 풀링 된 샘플 3 300-600 NT의 해상도를 할 수 있습니다. 많은 96 샘플 12 연속 CE가 실행 (그림 2)의 과정을 통해 분별을 준비 할 수있는 동안, 동시에 분별 할 수 있습니다 최대 반응의 8 세트까지. 또한, CEQ와 다른 유전자 분석기에서 나오는 데이터를 처리하고 분석하기 위해 개발 SHAPEfinder 소프트웨어는 더 자동화 및 SAFA 13이나 젤 분석 패키지보다 훨씬 적은 사용자 개입이 필요합니다. 고급 높은 처리량 방법은 최근에 이러한 얻기 위해 기술을 시퀀싱 다음 세대와 함께 구조 특정 효소보다는 알킬화 시약을 사용 PARS (RNA 구조의 병렬 분석) 14 수류탄 - 시퀀서 (조각 시퀀싱) 15로 등장 정 보RNA 구조에 대한 N. 이러한 기술의 매력에도 불구하고, 프로브 핵산에 내재 많은 제한은 여전히 16 남아있다. 이러한 문제는 차세대 시퀀싱은 기존의 모양에서 수행하는 것과 유사한 방식으로 화학적 변형과의 RNA 역전사 앞에는 SHAPE 시퀀싱 (SHAPE-시퀀서) 17 프로토콜에서 회피 할 수있다. 이러한 방법은 RNA 구조 결정의 미래를 나타낼 수 있지만, 그것은 차세대 시퀀싱은 매우 고가이며, 많은 실험실에 사용할 수없는 상태 것을 기억하는 것이 중요합니다. 셰이프 데이터 분석 유전자 분석기에서 생산 데이터를 마이그레이션 시간의 인덱스에 그려지는 모세관 검출기를 통과하는 시료의 형광 강도 (들)을 상기, electropherogram의 형태로 제공됩니다. 이 그래프는 네 개의 형광 채널에 해당하는 중복 흔적의 형태를 취s는 서로 다른 형광 물질을 감지하는 데 사용되며, 각 추적은 개별 cDNA를 또는 시퀀싱 제품에 해당하는 봉우리 구성입니다. Electropherogram 데이터는 탭으로 구분 된 텍스트 파일로 유전자 분석기에서 보낸 ShapeFinder 변환 및 분석 소프트웨어를 18로 가져옵니다. ShapeFinder는 처음 마이그레이션 시간과 최대 볼륨이 정확하게 각각 반응 생성물의 ID 및 수량을 반영하도록 데이터를 수학적 일련의 변환을 수행하는 데 사용됩니다. 피크는 다음 정렬 및 통합, 결과는 기본 RNA 시퀀스와 함께 표로 있습니다. RNA의 관련 세그먼트에 대한 &quot;반응 프로파일은&quot;(+) 염기 각 RNA와 관련된 값과 제어 값을 빼서, 아래 설명 된대로 데이터를 정규화하여 얻을 수 있습니다. 이 프로필은 정규화 된 반응성 발을 변환 (V5.3) 소프트웨어 19,20에게, RNAstructure로 가져RNA 이차 구조의 접이식 알고리즘에 통합 의사 에너지 제약에 단말. 이 방법으로 알고리즘을 프로빙 및 접는 화학 물질을 결합하여 현저하게 혼자 12,21 두 방법에 비해 구조 예측의 정확성을 향상시킵니다. RNAstructure의 출력 (V5.3)은 낮은 에너지의 이미지가 포함되어 RNA 이차 구조의 텍스트 점 괄호 표기법 SHAPE 반응성 프로파일 (들)뿐만 아니라 동일한 구조를 색으로 구분. 후자는 이후 같은 바르나 22 PseudoViewer 23와 같은 RNA 이차 구조의 그래픽 디스플레이 전용 소프트웨어로 내보낼 수 있습니다. <img alt="그림 1" fo:content-width="4.8in" fo:src="/files/ftp_upload/50243/50243fig1highres.jpg" src="/files/ftp_upload/50243/50243fig1.jpg" /> 그림 1. SHAPE 4,10를 통해 RNA의 구조 결정의 흐름도. (A) RNA M바깥은 생물 학적 샘플이나 시험 관내 전사하여 얻을 수. (B) 소스에 따라, RNA는 접하거나 처리 SHAPE 시약으로 변경됩니다. (C) 역 전사 형광이나 방사성 표시 프라이머를 사용하여. (D)의 cDNA 제품은 하나 모세관 또는 슬래브 겔 기반의 전기를 통해 분별. (E) 조각 분석. (F) RNA 구조 예측. <a href="http://www.jove.com/files/ftp_upload/50243/50243fig1large.jpg" target="_blank">큰 그림을 보려면 여기를 클릭하십시오.</a> <img alt="그림 2" fo:content-width="6in" fo:src="/files/ftp_upload/50243/50243fig2highres.jpg" src="/files/ftp_upload/50243/50243fig2.jpg" /> 그림 2. CE 기반 SHAPE의 높은 처리량 문자 신속한 여러 RNA를 분석, 및 / 또는 동일한 RNA의 여러 세그먼트를 할 수 있습니다. (A) </st룽&gt;는 RNA가 300-600 NT 섹션 (색, 녹색, 파란색과 빨간색으로 구분) (B) RNA의 섹션은 형광 프라이머의 집합을 (검은 색 화살표)를 사용하여 독립적으로 조사됩니다 (C)의 집합으로 나눌 수 있습니다 방법을 나타냅니다 반응은 ~ 3킬로바이트 RNA1에 대한 완전한 범위를 제공, 각각 우물 A1, B1, C1, 등으로 풀링 및로드됩니다. RNA를 2, 3, 4 등의 반응 제품은 유사하게 연속 전기 영동 실행의 분별을 준비 할 수 있습니다. <a href="http://www.jove.com/files/ftp_upload/50243/50243fig2large.jpg" target="_blank">큰 그림을 보려면 여기를 클릭하십시오.</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

생물 학적 과정에 관여 RNA의 기능을 이해하는 것은 RNA 구조의 철저한 지식이 필요합니다. 이를 위해, 불리는 방법은, 또는 모양 &quot;프라이머 확장하여 분석 높은 처리량의 선택 2 &#39;수산기 실화는&quot;단일 염기 해상도를 가진 RNA 이차 구조 예측을 할 수 있습니다. 이 방법은 우선적으로 수용액 RNA의 단일 좌초 또는 유연한 지역 acylate 화학 프로빙 에이전트를 사용합니다. 화학적 변형의 사이트는 수정 된 RNA의 역전사에 의해 감지하고,이 반응의 제품은 자동화 된 모세관 전기 영동 (CE)에 의해 분별입니다 수 있습니다. 역전사 효소는 SHAPE 시약에 의해 수정하는 RNA의 뉴클레오티드에서 일시 정지하기 때문에, 결과의 cDNA 라이브러리는 간접적으로 하나의 접힌 RNA의 맥락에서 좌초하는 리보 뉴클레오타이드를 매핑합니다. ShapeFinder 소프트웨어를 사용하여 자동화 CE에 의해 생성 된 electropherograms가 처리되고 뉴로 변환cleotide 반응성 테이블은 그 자체가 RNAStructure (V5.3) 예측 알고리즘에 사용 된 의사 에너지 제약 조건으로 변환됩니다. 실리코 RNA 이차 구조 예측에서와 프로브 형상을 결합하여 얻은 두 가지 차원 RNA 구조는 혼자 두 방법을 사용하여 얻은 구조보다 훨씬 더 정확한 것으로 판명되었다.

RNA가 존재 HIV-1 회전 반응 요소 (RRE)과는 3 &#39;말단 구조 카세트 4가 37 난방, 냉각, 및 배양 접힌 된 후, 관내 전사에 의해 선형화 된 플라스미드의 제조 하였다 ° C를 포함 MgCl 2. RNA는 NMIA에 노출 된 후 3 &#39;말단 구조 카세트에 하이브리드 말단 표지 된 DNA 프라이머로부터 역전사. 결과 모양의 cDNA 라이브러리를 함께 제어 및 시퀀싱 반응으로, 다음 그림 4에 나와 e...

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

프라이머 확장 (SHAPE)에 의해 분석 높은 처리량의 선택 2 &#39;수산기 실화는 단일 염기 해상도에서 수천 개의 뉴클레오티드로 수백에서의 RNA 구조를 결정하는 기술을 프로빙 소설 화학, 역전사, 모세관 전기 영동 및 이차 구조 예측 소프트웨어를 사용합니다.

높은 처리량 SHAPE를 사용하여 RNA 이차 구조 예측

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

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31.2K 조회수.  Frederick National Laboratory for Cancer Research. 프라이머 확장 (SHAPE)에 의해 분석 높은 처리량의 선택 2 '수산기 실화는 단일 염기 해상도에서 수천 개의 뉴클레오티드로 수백에서의 RNA 구조를 결정하는 기술을 프로빙 소설 화학, 역전사, 모세관 전기 영동 및 이차 구조 예측 소프트웨어를 사용합니다.

비디오: 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.

높은 처리량 효모 플라스미드 Overexpression 화면

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

연구

생물학

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.

컴퓨터 기반의 단백질 구조 및 기능 예측을위한 프로토콜

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.

Lipidic Bicelle 방법을 사용하여 막 단백질의 높은 처리량 결정

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.

자동을 사용하여 염색체의 높은 처리량 물리적 매핑<em&gt; 현장에서</em&gt; 하이브리드화

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.

집에서 만든 듀얼 글로우 루시 페라 제 분석 실험을 사용하여 높은 처리량 기능 검사

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.

Zebrafish의 배아를 사용하여 높은 처리량 접근 수동 작은 분자 화면

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.

높은 처리량 시퀀싱에 의한 마이크로 RNA 정량을위한 작은 RNA 분자의 고효율 내고

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.

후 전사적으로 규제 유전자의 화학 변조기에 대한 높은 처리량 검사

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;LIFE의 분석을 사용하여 높은 처리량 miRNA의 표적의 탐지

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.

높은 처리량 검열을 사용 키나제 기판 쌍의 식별


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