이 작품은 건강 RO1 - GM085130 국립 연구소와 국립 과학 재단 (National Science Foundation) MCB0929394에서 보조금에 의해 지원되었다. 우리는 그녀의 환대와 그녀의 실험실에서 영화 우리에게 허용에 대한 박사 메리언 슈미트 감사합니다.

1. Footprinting의 시약의 준비 <ol><li> 100 MM 나트륨 cacodylate, 1 ㎜ EDTA (에틸렌 다이아 민 테트라 초산), 1 M KCl을 포함 10X 반응 버퍼를 준비합니다. 7.4로 산도를 조정합니다. 0.2 μm의 아세테이트 필터 장치 (Nalgene)를 사용하여 버퍼를 필터링합니다. 비고가 : 10X 버퍼에 직접 pipet의 RNA를하지 않습니다. </li><li> 로 표 1에 표시된 각 반응에 대한 적정 반응 믹스를 준비합니다. 적정 믹스의 볼륨 (원하는 농도에 1X 버퍼와 MG (II)는) 1X 버퍼에서 RNA의 10μl를 추가하기 전에, 90 μl해야합니다. </li><li> 6.63M 요소, 20mM 나트륨 구연산, EDTA (에틸렌 다이아 민 테트라 초산) 1mM, 0.25 μg / μl tRNA, 0.025 %의 크실렌의 cyanol, 그리고 0.025 %의 bromphenol 블루를 포함하는 RNAse T1의 소화 버퍼를 준비합니다. 이 버퍼는 최대 6 개월까지 4 ° C에 저장할 수 있습니다. </li><li> 펜톤 반응은 250 MM 철 (NH 4) 2 (SO 4) 2, 250 MM EDTA (에틸렌 다이아 민 테트라 초산), 500 MM 나트륨 L - 아스코 브 산의 신선한 수성 솔루션을 필요로합니다. 농도는 제 1에 명시된0.5은 래디 칼의 다양한 이가의 이온 농도를 청소하지 않는 등 나트륨 cacodylate로 버퍼에 평형 실험에 최적입니다. </li><li> peroxidative (1.5.1)과 산화 (1.5.2) footprinting 반응에 대한 시약. <ol><li> peroxidative 펜톤 반응은 100 MM 철 - EDTA (에틸렌 다이아 민 테트라 초산) 솔루션, 50 MM 나트륨 L - 아스코 브 산 및 1.5 % H 2 O 2의 신선한 수성 솔루션을 필요로합니다. 철 - EDTA (에틸렌 다이아 민 테트라 초산) 솔루션은 철분 이상의 EDTA (에틸렌 다이아 민 테트라 초산) 1.1 배 초과를 가질 EDTA (에틸렌 다이아 민 테트라 초산)과 혼합 철 (NH 4) 2 (SO 4) 2 준비가되어 있습니다. </li><li> 그냥 산화 절단 반응 (3.4.2)를 시작하기 전에 μl 철 2 μl (NH 4) 2 (SO 4) 2 (250 ㎜), EDTA (에틸렌 다이아 민 테트라 초산) 2.2 μl (250 ㎜), 62.5를 결합하여 펜톤 반응 믹스를 만들 나트륨 L - 아스코 브 산 (500 ㎜), 22.5 μl 반응 10X 버퍼와 0 footprinting 반응 혼합물 (3.4.2)에 최종 농도에 도달하는 H 2 O의 135.5 μl.10 MM, 0.11 MM, 그리고 6.61 MM, 각각. </li></ol></li></ol> 2. Footprinting 실험을위한 RNA 준비 <ol><li> 짧은 50 세포핵 (DNA 통합 기술, www.idtdna.com에서 예) 구입할 수있다면 RNA는 DNA 템플릿 6 시험 관내 전사의 표준에 의해 생산 또는 수 있습니다. footprinting 실험 중에 RNA의 샘플 무결성을 유지하는 것이 좋습니다 실험실 청결 절차를 따르십시오. 7 </li><li> 체외 베꼈는데 RNA에의 Dephosphorylation 8 이전 RNA의 5 &#39;끝에 32 P 라벨이 필요합니다. RNA 10 pmol는 kinasing에 의해 최종 분류됩니다. 9 솔루션은 인산 가수 분해 효소의 불활 성화 성공 후 뿌연한다. </li><li> denaturing 겔 전기 영동을 사용하여 radioactively 레이블 RNA 정화. RNA는 0.3 M의 아세트산 나트륨 (산도 5.5) 사용하여 두 후속 젤 extractions하여 복구할 수 있습니다. RNA는 1 ML의 에탄올과 이후로 0.5 ML 수용액을 혼합하여 시켰던 것입니다드라이 아이스 1 분간 인큐베이션. 4 ° C, 0.5 시간을 위해 12,500 RPM에서 샘플을 돌린다. 뜨는을 취소하고 70 % 에탄올로 RNA 펠렛을 씻는다. 추가 원심 분리 후 뜨는 제거하고 진공에서 펠렛을 건조. </li><li> 반응 관의 RNA 솔루션, 에탄올과 진공 건조 RNA와 침전물의 1X Buffer.Pipet 30 μl의 330 UL에서 32 P - 표시 RNA 샘플을 분해, 참조 사다리 (단계 3.5) 생성이 튜브를 유지. 95에서 가열하여 버퍼 RNA 솔루션 ° 2 분 C를 변성. 15 분 상온에서 샘플을 냉정하고 다시 솔루션에 튜브의 뚜껑에 condensates을 가지고 빠른 스핀을 제공합니다. </li></ol> 3. Footprinting 실험 <ol><li> 전기 장식은 분석 젤 당 데이터 요소의 최대 개수를 결정합니다. 우리는 30 잘 빗을 사용합니다. 참조 사다리 2 우물과 uncleaved 컨트롤 하나를 가득 메우고 있습니다. 27 반응 튜브를 준비합니다. 최종 MG (II) concentrati기능은 고르게 적정의 midpoint 주위 규모의 주문을 걸쳐 로그 규모 간격해야합니다. </li><li> 별도로, 5 분 50 RNA와 MG (II) 솔루션 ° C를 품어. 100 μl의 최종 볼륨을 도달 1X 버퍼에 MG의 해당 금액 (II) 90 μl와 RNA 용액 10 μl를 섞는다. 50 30 분 솔루션을 품어 ° C. </li><li> 접는가 발생하는 동안 솔루션은 25 ° C 1 시간에 대한 평형하자. </li><li> Peroxidative (3.4.1) 또는 산화 (3.4.2) 수산기 라디칼 footprinting 반응. <ol><li> 2 μl 철 - EDTA (에틸렌 다이아 민 테트라 초산) (100 ㎜)의 방울을 배치하여 peroxidative 반응을 시작, 2 μl 나트륨 L - 아스코 브 산 (50 ㎜) 2 μl H 2 O 2 (1.5 %)는 반응의 맨 안쪽에 서로 구분 튜브는 RNA 솔루션을 포함하고 활발한 혼합하여 footprinting 반응을 시작합니다. 300 μl 감기 절대 에탄올을 추가로 약 15 초 후에 반응을 중지합니다. 튜브에게 3-5 번 돌립니다. 침전, 세척 및2.4 단계로 펠렛을 건조. </li><li> 신선한 펜톤 반응 믹스 (1.6) 5 μl을 추가하여 산화 히드록실 급진 footprintingreaction를 시작합니다. 25 30 분 품어 ° C. 튜브에게 3-5 번 회전하여 반응과 혼합을 담금질하기 위해 추운 절대 에탄올 300 μl를 추가합니다. 침전, 세척 및 펠렛대로 단계 2.4 표시된 건조. </li></ol></li><li> 단계 1.3 준비 솔루션을 사용하여 표준 절차에 따라 10 RNase T1 다이제스트 참조 예제를 생성합니다. </li><li> 8 μl 젤로드 염료 II (AMBION)와 확인 RNA에서 RNA의 알약을 분해은 가이거 계수기를 사용하여 resuspended 있습니다. </li><li> 11.로드 샘플이 참조 및 제어를 포함하여 표준 프로토콜에 따라 denaturing, 8 % polyacrylamide의 순서 젤을 준비합니다. 60 W에서 별도의 RNA 조각 - 2.5 시간에 대해 75 W. 하룻밤 저장 형광체 화면으로 건조 겔을 쉽게받을 수 있습니다. filmless autoradiography 예위한 이미징 시스템 스캔 형광체 화면. 폭풍 865 (GE 헬스케어) 또는 태풍 (GE 헬스케어). 컴퓨터에 겔 이미지 파일을 전송합니다. </li></ol> 4. 데이터 분석 <ol><li> 각 염기의 수산기 라디칼 반응은 분석 젤의 각 밴드의 강도를 quantifying에 의해 결정됩니다. 다운로드, 설치 및 오픈 사파, 단일 밴드 피팅 및 부량을위한 오픈 소스 소프트웨어를 사용하기 편리합니다. 12, 13 사용 설명서의 지침을 따르십시오. 간단히 같은 RNA 시퀀스를로드합니다. txt 파일은. 겔 파일로 겔 사진으로 따라갔다. 밴드 농도를 조절하고, 차선을 정의 앵커 차선을 선택하고 젤 정렬을 수행합니다. 세포핵에 밴드의 임무는 RNase T1의 소화 사다리를 참조에서 발생합니다. 밴드 integrals를 정할 가능성이 불변량 잔류물을 정상화하고 할당 정상화 / colorplot 기능을 사용합니다. . txt 파일과 같은 출력을 저장합니다. </li><li> 사파가에서 대표 겔에 골목길과 행을 대표하는 열을 포함하는 스프레드 시트를 출력RNA의 조각에 해당하는 각각의 밴드의 밴드 밀도를 tegrated. 첫째, 끝점 MG (II) 농도 (예제 50 ㎜)의 프로필과 함께 MG (II) 샘플 노에서 파생 보호 프로파일을 비교하여 용매 접근에 눈에 띄는 변화를 표시 보호 사이트를 식별합니다. 낮은 값을, 더 뉴클레오 티드가 히드록실 래디 칼과 반대의 공격으로부터 보호합니다. </li><li> 개인 또는 MG (II) 농도 간의 세포핵의 그룹의 표준 대역 integrals에서 접는 등온선을 만듭니다. •을 - 등온선은 개별적 - Fi 인터넷 접속 = L + (L U)에 의해 부분 채도로 결정됩니다 <img src="files/ftp_upload/3244/3244y.jpg" alt="Y" /> F는 밴드의 통합 밀도 (들)을 나타냅니다 어디에 분석하고, L과 U는 전환 아래쪽과 위쪽 한계를 나타내며 <img src="/files/ftp_upload/3244/3244y.jpg" alt="Y" /> 분수 채도입니다. 14 데이터가 적합한 비선형 리터를 사용하는힐 방정식 (1) 동쪽 평방 분석 프로그램 (우리도 마찬가지 유래 (OriginLab) 또는 GraphPad (GraphPad 소프트웨어, 주식 회사)를 사용합니다.) <img alt="등식 1" src="/files/ftp_upload/3244/3244eq1.jpg" /> K D의 평형이 어디 해리 상수, [M] 접는 반응을 mediates 변수,이 예제에서 MG (II)의 농도에 해당하고, N H는 힐 계수이다. 이 절차는 비늘 부분 포화로의 전환은 그 midpoint을 결정하고 전환이 sigmoidal 여부의 현상 학적 테스트를 제공합니다. 사파 (그림 3B)에 의해 분석하고 위에 설명된대로 맞는 순서 젤 (그림 3A)에서 파생된 등온선은 그림 3C에 표시됩니다. 그림 3에 표시된 부분 포화 등온선은 스프레드 시트에서 스케일링에 의해 생성되었다 방정식 분율을 사용하여 최고 맞게 위턱과 아래턱에 한계 (U와 L)에 대한 사파에 의해 생성된 값을 = 값 / (U - L) - L / (U - L). </li></ol> 5. 대표 결과 : 그림 3은 P4 - P6 RNA • OH의 footprinting 실험에서 담당자 결과를 보여줍니다. 겔 이미지 (왼쪽) 단일 염기 과제로 인해 T1 차선 (각 G의 RNase T1의 앞을)에서 잘 정의된 밴드 가능합니다)) 배경 절단은 (오른쪽 차선) B 최소한의 것을 나타냅니다하고, C) 수산기 라디칼 RNA의 유도 조각은 잘 배경 위에있다. 높은 MG (II)로 낮은에서 전환은 개인 및 RNA 차 구조의 형성을 나타내는 밴드 그룹의 통합 밴드 밀도를 감소와 연관되어있다. 단일 또는 그의 절단 변경 concomitantly &#39;보호&#39;라고 인접한 뉴클레오 티드 그룹. 밀접한의 결정 구조에서 관찰 분자의 용매 액세스할 지역에 해당하는 MG (II)의 특징 • OH 보호는 P4 - P6 RNA의 접는을 중재. 15 접힌 RNA 분자 C<html> 분명 구조적 또는 동적 현상과 상관되지 않은 차 연락 보호 매우 다른 extents (즉, 어떻게 배경 밴드 밀도 감소를 닫습니다)가. 따라서 일부 RNA 분자는 그림 3과 같이, 잘 정의된 구조 전환을 표시되며, 몇몇은하지 않습니다. 밴드 농도는 사파 12 분석 (그림 3B)에 의해 계량 및 표준입니다. 출력 • OH에 대한 보호의 상대 정도를 시각화 &#39;열&#39;플롯이다. 컬러 전환은 MG (II) 또한시 접근의 변화에​​ 대해 설명합니다. 각각 빨간색 또는 파란색 보여줍 더 접근 이상의 세포핵 보호에 화이트. 음영의 각 학위는 보호 곡선 (그림 3C)로 꾸몄다과 힐 방정식 (1)과 같은 바인딩 모델을 분석할 수 수치와 연관되어있다. 지속적인 분리가 제한 153-155 부속 평형 보호 163-164의 해당 가치의 약 두배입니다. ontent &quot;&gt; <img alt="그림 1" src="/files/ftp_upload/3244/3244fig1.jpg" /> 그림 1. 수산기 라디칼 생산과 P4 - P6 RNA를 접는. A) 철 (II)는 과산화수소의 히드록실 래디 칼의 생성을 catalyzes. 아스코 브 산이 함유 철에 철 (III) 다시 줄어 듭니다. B) MG (II)의 부재에서 더 P4 - P6 차 구조는 히드록실 래디 칼은 모든 접근 백본 위치를 침투하고 다니엘 있도록 형성되지 않습니다. MG의 추가 (II)는 오직 용매 접근 백본은 히드록실 래디 칼에 의해 죽습 수 있도록, P4 - P6의 폴딩 시작합니다. <img alt="그림 2" src="/files/ftp_upload/3244/3244fig2.jpg" /> 그림 2. 히드록실 급진적인 footprinting 실험에 대한 개요. A) Dephophorylation와 RNA의 32 P 5&#39; 엔드 라벨. B) 32 정제는 denaturing polyacrylamide 젤에서 RNA를 P - 레이블. C) RNA 밴드의 절단, 후속 RNA 추출, 에탄올 침전. D) Prefolding과RNA의 접는. E) 갓 준비 펜톤 반응 혼합물의 추가는 히드록실 래디 칼을 생성합니다. polyacrylamide 겔 전기 영동을 denaturing하여 F) RNA 조각 분리. 사파 소프트웨어에 의해 RNA 조각의 G) Quantitation. <img alt="그림 3" src="/files/ftp_upload/3244/3244fig3.jpg" /> 그림 3. peroxidative 펜톤의 footprinting 반응 후 RNA 조각의 분석. (A) RNA는 히드록실 래디 칼에 노출되었고 절단 제품은 denaturing polyacrylamide 겔 전기 영동 (PAGE)를 사용하여 해결이되었습니다. 사진은 MG의 농도 증가 (II)와 peroxidative 절단 제품을 보여줍니다. 참조 및 제어 골목길이 표시됩니다. 이 이미지 파일은 각각의 밴드의 볼륨을 quantitates 사파 프로그램에 입력됩니다. (B) &#39;열&#39;줄거리는 사파에 의해 생성된. (C) 사파에서 염기 번호를 출력 통합 밴드 밀도와 관련된 가치의 스프레드 시트는 같은 힐 equati으로 바인딩 모델로 분석됩니다에 (1). 보호 지역은 MG (II) 농도의 함수로 밴드 통합 밀도의 증가에 따라 선정되었습니다. K D는 RNA의 절반이 모니터링되고있는 사이트에 접혀있는의 MG (II) 농도입니다. 힐 계수, N H은 구속력 cooperativity에 대한 정보를 제공하고이 특정 사이트의 접이식에 관련된 마그네슘 이온의 개수에 대한 낮은 견적을 제공 곡선의 기울기의 측정이다. <img alt="표 1" src="/files/ftp_upload/3244/3244table1.jpg" /> 표 1. 다른 MG (II) 농도를 포함하는 RNA 샘플을 생성하는 대표적인 볼륨.

<ol>
	<li>Tullius, T. D., Dombroski, B. A. <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":+high-resolution+information+about+DNA-protein+contacts+and+application+to+lambda+repressor+and+Cro+protein.">Hydroxyl radical "footprinting": high-resolution information about DNA-protein contacts and application to lambda repressor and Cro protein.</a> Proc. Natl. Acad. Sci. U.S.A. 83, 5469-5473 (1986).</li><li>Celander, D. W., Cech, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iron(II)-ethylenediaminetetraacetic+acid+catalyzed+cleavage+of+RNA+and+DNA+oligonucleotides:+similar+reactivity+toward+single-+and+double-stranded+forms.">Iron(II)-ethylenediaminetetraacetic acid catalyzed cleavage of RNA and DNA oligonucleotides: similar reactivity toward single- and double-stranded forms.</a> Biochemistry. 29, 1355-1361 (1990).</li><li>Celander, D. W., Cech, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+the+higher+order+folding+of+a+catalytic+RNA+molecule.">Visualizing the higher order folding of a catalytic RNA molecule.</a> Science. 251, 401-407 (1991).</li><li>Sclavi, B., Sullivan, M., Chance, M. R., Brenowitz, M., Woodson, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+folding+at+millisecond+intervals+by+synchrotron+hydroxyl+radical+footprinting.">RNA folding at millisecond intervals by synchrotron hydroxyl radical footprinting.</a> Science. 279, 1940-1943 (1998).</li><li>Tullius, T. D., Dombroski, B. A., Churchill, M., Kam, L. <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:+a+high-resolution+method+for+mapping+protein-DNA+contacts.">Hydroxyl radical footprinting: a high-resolution method for mapping protein-DNA contacts.</a> Methods. Enzym. 155, 537-558 (1987).</li><li>Milligan, J. F., Groebe, D. R., Witherell, G. W., Uhlenbeck, O. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligoribonucleotide+synthesis+using+T7+RNA+polymerase+and+synthetic+DNA+templates.">Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates.</a> Nucleic. Acids. Res. 15, 8783-8798 (1987).</li><li>Schlatterer, J. C., Brenowitz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complementing+global+measures+of+RNA+folding+with+local+reports+of+backbone+solvent+accessibility+by+time+resolved+hydroxyl+radical+footprinting.">Complementing global measures of RNA folding with local reports of backbone solvent accessibility by time resolved hydroxyl radical footprinting.</a> Methods. 49, 142-147 (2009).</li><li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cloning: A Laboratory Manual. , (2001).</li><li>Zaug, A. J., Grosshans, C. A., Cech, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequence-specific+endoribonuclease+activity+of+the+Tetrahymena+ribozyme:+enhanced+cleavage+of+certain+oligonucleotide+substrates+that+form+mismatched+ribozyme-substrate+complexes.">Sequence-specific endoribonuclease activity of the Tetrahymena ribozyme: enhanced cleavage of certain oligonucleotide substrates that form mismatched ribozyme-substrate complexes.</a> Biochemistry. 27, 8924-8931 (1988).</li><li>Knapp, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+approaches+to+probing+of+RNA+secondary+and+tertiary+structure.">Enzymatic approaches to probing of RNA secondary and tertiary structure.</a> Methods. Enzym. 180, 192-212 (1989).</li><li>Slatko, B. E., Albright, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Denaturing+gel+electrophoresis+for+sequencing.">Denaturing gel electrophoresis for sequencing.</a> Curr. Protoc. Mol. Biol. Chapter 7, Unit 7.6-Unit 7.6 (2001).</li><li>Das, R., Laederach, A., Pearlman, S. M., 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=SAFA:+semi-automated+footprinting+analysis+software+for+high-throughput+quantification+of+nucleic+acid+footprinting+experiments.">SAFA: semi-automated footprinting analysis software for high-throughput quantification of nucleic acid footprinting experiments.</a> RNA. 11, 344-354 (2005).</li><li>Simmons, K., Martin, J. S., Shcherbakova, I., Laederach, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+quantification+and+analysis+of+kinetic+*OH+radical+footprinting+data+using+SAFA.">Rapid quantification and analysis of kinetic *OH radical footprinting data using SAFA.</a> Methods. Enzym. 468, 47-66 (2009).</li><li>Uchida, T., He, Q., Ralston, C. Y., Brenowitz, M., Chance, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linkage+of+monovalent+and+divalent+ion+binding+in+the+folding+of+the+P4-P6+domain+of+the+Tetrahymena+ribozyme.">Linkage of monovalent and divalent ion binding in the folding of the P4-P6 domain of the Tetrahymena ribozyme.</a> Biochemistry. 41, 5799-5806 (2002).</li><li>Cate, J. H., Gooding, A. R., Podell, E., Zhou, K., Golden, B. L., Kundrot, C. E., Cech, T. R., Doudna, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+a+group+I+ribozyme+domain:+principles+of+RNA+packing.">Crystal structure of a group I ribozyme domain: principles of RNA packing.</a> Science. 273, 1678-1685 (1996).</li><li>Petri, V., Brenowitz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+nucleic+acids+footprinting:+thermodynamic+and+kinetic+approaches.">Quantitative nucleic acids footprinting: thermodynamic and kinetic approaches.</a> Curr. Opin. Biotechnol. 8, 36-44 (1997).</li><li>Takamoto, K., Das, R., He, Q., Doniach, S., Brenowitz, M., Herschlag, D., Chance, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+RNA+compaction:+insights+from+the+equilibrium+folding+pathway+of+the+P4-P6+RNA+domain+in+monovalent+cations.">Principles of RNA compaction: insights from the equilibrium folding pathway of the P4-P6 RNA domain in monovalent cations.</a> J. Mol. Biol. 343, 1195-1206 (2004).</li><li>Brenowitz, M., Senear, D. F., Shea, M. A., Ackers, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+DNase+footprint+titration:+a+method+for+studying+protein-DNA+interactions.">Quantitative DNase footprint titration: a method for studying protein-DNA interactions.</a> Methods. 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>

관심 없음 충돌 선언하지 않습니다.

히드록실 급진 footprinting는 핵산의 용매 접근 표면적을 평가하는 유용한 도구입니다. 차 구조 14 질적 및 양적 형성은 이러한 이온 종류와 농도, 산도, 온도, 바인딩 단백질이나 접는 공동 요인으로 매개 변수의 함수로 다음 수 있습니다. 정직하고 저렴한 프로토콜과 그 결과 용매 접근성 및 단일 뉴클레오 티드 수준에서 접는 정보의 강력한 조합이 방법은 매우 매력적합니다. 전통적 • OH?...

<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

RNA의 분자 생물학에서 필수적인 역할을한다. 유전 정보를 전달하는 이외에, RNA는 레귤레이터, 바인더 또는 촉매와 같은 특정 생물 학적 역할을 수행 고유한 삼차 구조로 접을 수 있습니다. 차 접촉 형성에 관한 정보는 RNA 분자의 기능을 이해하는 것이 필수적입니다. 히드록실 래디 칼 (• OH)가 높은 반응과 작은 크기로 인해 핵산의 구조 독특한 프로브입니다. 1 footprinting 프로브로 사용하면, 히드록실 래디 칼과 함께 DNA 1과 RNA 2의 phosphodiester 백본의 용매 접근 표면을지도 단일 뉴클레오 티드 해상도만큼 괜찮아요. 히드록실 라디칼 footprinting는 DNA - 단백질 1과 RNA - 단백질 단지의 예 분자간 접촉면 내에 세포핵을 식별하는 데 사용할 수 있습니다. 평형 3 번과 4 번 전환 운동은 soluti의 함수로 수산기 라디칼 footprinting을 실시하여 확인할 수 있습니다에서 변수 또는 시간을 각각. footprinting의 주요 기능은 프로브에게 노출 제한 (예 : &#39;싱글 히트 속도론&#39;) 폴리머의 각 염기의 유니폼을 샘플링 결과입니다 5. 이 비디오 문서에서, 우리는 RNA 샘플의 준비와 MG (II) - 중재 접는 등온선의 결정을 설명하기 Tetrahymena ribozyme의 P4 - P6 도메인을 사용합니다. 우리는 H 2 O 2 (우리가이 &#39;peroxidative&#39;프로토콜 호출)와 자연스럽게 녹아 O 2를 사용하는 귀중한하지만, 널리 알려진 아니라, 대안을 (우리가 부르는 &#39;가 필요합니다 잘 알려진 수산기 라디칼 footprinting 프로토콜의 사용을 설명 산화 &#39;프로토콜). 데이터 감소, 변환 및 분석 절차의 개요가 제공됩니다.

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

이 프로토콜은 MG (II) - 종속 히드록실 급진 footprinting 두 가지 방법으로 RNA 차 구조 형성을 계량하는 방법을 설명합니다.

&#39;Peroxidative&#39;와 &#39;산화&#39;히드록실 급진적 Footprinting에 의해 RNA 구조의 모니터링 평형 변경

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

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Biology

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

14.2K 조회수.  Hunter College. 이 프로토콜은 MG (II) - 종속 히드록실 급진 footprinting 두 가지 방법으로 RNA 차 구조 형성을 계량하는 방법을 설명합니다.

비디오: 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.

이중층 탄성 등록 정보 모니터링 변경에 대한 단일 분자 방법

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

연구

생물학

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.

유전자 변형으로 인코딩된 칼슘 센서를 사용하여 Apoptosis 동안 Mitochondrial 칼슘 레벨에서 동적 변경 사항 모니터링

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 세포 Transfection 얹는 Gaussia의 루시페라제의 mRNA의 시험 관내 스크립트 작성 및 상한에

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.

갑각류와 장기 치명적인 독성 시험 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 정제에 의한 염색질 절연 (처프)

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.

형광에 의한 바이러스성 RNA의 탐지<em&gt; 현장에서</em&gt; 하이브리드화 (물고기)

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.

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 ...

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.

정제 된 야생형과 돌연변이 체 CFTR 단백질의 기능의 재구성 및 채널 활동 측정

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 분석에 의한 수용체 후 국제화 인신 매매에 약물 유발의 이동을 추적

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.

대변​​ 글루코 코르티코이드 분석 : equids는 비 침습적 부신 모니터링

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