Wir sind dankbar für die gemeinsame Nutzung Jiří Sponer die Koordinaten der DNA-Doppelhelix in Molekulardynamik-Simulationen erzeugt. Wir erkennen auch Nada Spackova um Hilfe beim Herunterladen dieser Strukturen. Unterstützung dieser Arbeit durch USPHS Forschungsstipendien GM34809 und GM096889 gedankt.

<ol>
	<li>Lu, X. -. J., Olson, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3DNA:+a+software+package+for+the+analysis,+rebuilding,+and+visualization+of+three-dimensional+nucleic+acid+structures.">3DNA: a software package for the analysis, rebuilding, and visualization of three-dimensional nucleic acid structures.</a> Nucleic Acids Res. 31, 5108-5121 (2003).</li><li>Lu, X. -. J., Olson, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3DNA:+a+versatile,+integrated+software+system+for+the+analysis,+rebuilding,+and+visualization+of+three-dimensional+nucleic-acid+structures.">3DNA: a versatile, integrated software system for the analysis, rebuilding, and visualization of three-dimensional nucleic-acid structures.</a> Nature Protocols. 3, 1213-1227 (2008).</li><li>Zheng, G., Lu, X. -. J., Olson, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Web+3DNA-a+web+server+for+the+analysis,+reconstruction,+and+visualization+of+three-dimensional+nucleic-acid+structures.">Web 3DNA-a web server for the analysis, reconstruction, and visualization of three-dimensional nucleic-acid structures.</a> Nucleic Acids. Res. 37, W240-W246 (2009).</li><li>Xin, Y., Olson, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BPS:+a+database+of+RNA+base-pair+structures.">BPS: a database of RNA base-pair structures.</a> Nucleic Acids Res. 37, D83-D88 (2009).</li><li>Zheng, G., Colasanti, A. V., Lu, X. -. J., Olson, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3DNALandscapes:+a+database+for+exploring+the+conformational+features+of+DNA.">3DNALandscapes: a database for exploring the conformational features of DNA.</a> Nucleic Acids Res. 38, 267-274 (2010).</li><li>Tolstorukov, M. Y., Colasanti, A. V., McCandlish, D., Olson, W. K., Zhurkin, V. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+'roll-and-slide'+mechanism+of+DNA+folding+in+chromatin.+Implications+for+nucleosome+positioning.">A novel 'roll-and-slide' mechanism of DNA folding in chromatin. Implications for nucleosome positioning.</a> J. Mol. Biol. 371, 725-738 (2007).</li><li>Lu, X. -. J., Olson, W. K., Bussemaker, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RNA+backbone+plays+a+crucial+role+in+mediating+the+intrinsic+stability+of+the+GpU+dinucleotide+platform+and+the+GpUpA/GpA+miniduplex.">The RNA backbone plays a crucial role in mediating the intrinsic stability of the GpU dinucleotide platform and the GpUpA/GpA miniduplex.</a> Nucleic Acids Res. 38, 4868-4876 (2010).</li><li>Mouw, K. W., Rice, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shaping+the+Borrelia+burgdorferi+genome:+crystal+structure+and+binding+properties+of+the+DNA-bending+protein+Hbb.">Shaping the Borrelia burgdorferi genome: crystal structure and binding properties of the DNA-bending protein Hbb.</a> Mol. Microbiol. 63, 1319-1339 (2007).</li><li>Burgdorfer, W., Barbour, A. G., Hayes, S. F., Benach, J. L., Grunwaldt, E., Davis, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lyme+disease-a+tick-borne+spirochetosis?.">Lyme disease-a tick-borne spirochetosis?.</a> Science. 216, 1317-1319 (1982).</li><li>Benach, J. L., Bosler, E. M., Hanrahan, J. P., Coleman, J. L., Habicht, G. S., Bast, T. F., Cameron, D. J., Ziegler, J. L., Barbour, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spirochetes+isolated+from+the+blood+of+two+patients+with+Lyme+disease.">Spirochetes isolated from the blood of two patients with Lyme disease.</a> N. Engl. J. Med. 308, 740-742 (1983).</li><li>Lanka&#353;, F., &#352;pa&#269;kov&#225;, N., Moakher, M., Enkhbayar, P., &#352;poner, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+measure+of+bending+in+nucleic+acids+structures+applied+to+A-tract+DNA.">A measure of bending in nucleic acids structures applied to A-tract DNA.</a> Nucleic Acids Res. 38, 3414-3422 (2010).</li><li>Romanuka, J., Folkers, G. E., Biris, N., Tishchenko, E., Wienk, H., Bonvin, A. M. J. J., Kaptein, R., Boelens, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specificity+and+affinity+of+Lac+repressor+for+the+auxiliary+operators+O2+and+O3+are+explained+by+the+structures+of+their+protein-DNA+complexes.">Specificity and affinity of Lac repressor for the auxiliary operators O2 and O3 are explained by the structures of their protein-DNA complexes.</a> J. Mol. Biol. 390, 478-489 (2009).</li><li>Berman, H. M., Westbrook, J., Feng, Z., Gilliland, G., Weissig, H., Shindyalov, I. N., Bourne, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Protein+Data+Bank.">The Protein Data Bank.</a> Nucleic Acids. Res. 28, 235-242 (2000).</li><li>Joint, I. U. P. A. C. -. I. U. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Commission+on+Biochemical+Nomenclature+(JCBN)+Abbreviations+and+symbols+for+the+description+of+conformations+of+polynucleotide+chains.">Commission on Biochemical Nomenclature (JCBN) Abbreviations and symbols for the description of conformations of polynucleotide chains.</a> Eur. J. Biochem. 131, 9-15 (1983).</li><li>Altona, C., Sundaralingam, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformational+analysis+of+the+sugar+ring+in+nucleosides+and+nucleotides.+A+new+description+using+the+concept+of+pseudorotation.">Conformational analysis of the sugar ring in nucleosides and nucleotides. A new description using the concept of pseudorotation.</a> J. Am. Chem. Soc. 94, 8205-8212 (1972).</li><li>Dickerson, R. E., Bansal, M., Calladine, C. R., Diekmann, S., Hunter, W. N., Kennard, O., von Kitzing, E., Lavery, R., Nelson, H. C. M., Olson, W. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Definitions+and+nomenclature+of+nucleic+acid+structure+parameters.">Definitions and nomenclature of nucleic acid structure parameters.</a> J. Mol. Biol. 205, 787-791 (1989).</li><li>Olson, W. K., Bansal, M., Burley, S. K., Dickerson, R. E., Gerstein, M., Harvey, S. C., Heinemann, U., Lu, X. -. J., Neidle, S., Shakked, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+standard+reference+frame+for+the+description+of+nucleic+acid+base-pair+geometry.">A standard reference frame for the description of nucleic acid base-pair geometry.</a> J. Mol. Biol. 313, 229-237 (2001).</li><li>Lavery, R., Moakher, M., Maddocks, J. H., Petkeviciute, D., Zakrzewska, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformational+analysis+of+nucleic+acids+revisited:+Curves+.">Conformational analysis of nucleic acids revisited: Curves+.</a> Nucleic Acids Res. 37, 5917-5929 (2009).</li><li>Franklin, R. E., Gosling, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+configuration+in+sodium+thymonucleate.">Molecular configuration in sodium thymonucleate.</a> Nature. 171, 740-741 (1953).</li><li>Watson, J. D., Crick, F. H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetical+implications+of+the+structure+of+deoxyribonucleic+acid.">Genetical implications of the structure of deoxyribonucleic acid.</a> Nature. 171, 964-967 (1953).</li><li>Marvin, D. A., Spencer, M., Wilkins, M. H. F., Hamilton, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+configuration+of+deoxyribonucleic+acid.">A new configuration of deoxyribonucleic acid.</a> Nature. 182, 387-388 (1958).</li><li>Berman, H. M., Olson, W. K., Beveridge, D. L., Westbrook, J., Gelbin, A., Demeny, T., Hsieh, S. -. H., Srinivasan, A. R., Schneider, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Nucleic+Acid+Database:+a+comprehensive+relational+database+of+three-dimensional+structures+of+nucleic+acids.">The Nucleic Acid Database: a comprehensive relational database of three-dimensional structures of nucleic acids.</a> Biophys. J. 63, 751-759 (1992).</li><li>Stella, S., Cascio, D., Johnson, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+shape+of+the+DNA+minor+groove+directs+binding+by+the+DNA-bending+protein+Fis.">The shape of the DNA minor groove directs binding by the DNA-bending protein Fis.</a> Genes Dev. 24, 814-826 (2010).</li><li>Swigon, D., Coleman, B. D., Olson, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+the+Lac+repressor-operator+assembly:+the+influence+of+DNA+looping+on+Lac+repressor+conformation.">Modeling the Lac repressor-operator assembly: the influence of DNA looping on Lac repressor conformation.</a> Proc. Natl. Acad. Sci. U.S.A. 103, 9879-9884 (2006).</li><li>Czapla, L., Swigon, D., Olson, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+the+nucleoid+protein+HU+on+the+structure,+flexibility,+and+ring-closure+properties+of+DNA+deduced+from+Monte-Carlo+simulations.">Effects of the nucleoid protein HU on the structure, flexibility, and ring-closure properties of DNA deduced from Monte-Carlo simulations.</a> J. Mol. Biol. 382, 353-370 (2008).</li><li>Czapla, L., Peters, J. P., Rueter, E. M., Olson, W. K., Maher, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+apparent+DNA+flexibility+enhancement+by+HU+and+HMGB+proteins:+experiment+and+simulation.">Understanding apparent DNA flexibility enhancement by HU and HMGB proteins: experiment and simulation.</a> J. Mol. Biol. 409, 278-289 (2011).</li><li>Auffinger, P., Hashem, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SwS:+a+solvation+web+service+for+nucleic+acids.">SwS: a solvation web service for nucleic acids.</a> Bioinformatics. 23, 1035-1037 (2007).</li><li>Dror, O., Nussinov, R., Wolfson, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ARTS+web+server+for+aligning+RNA+tertiary+structures.">The ARTS web server for aligning RNA tertiary structures.</a> Nucleic Acids Res. 34, 412-415 (2006).</li><li>Dixit, S. B., Beveridge, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+bioinformatics+of+DNA:+a+web-based+tool+for+the+analysis+of+molecular+dynamics+results+and+structure+prediction.">Structural bioinformatics of DNA: a web-based tool for the analysis of molecular dynamics results and structure prediction.</a> Bioinformatics. 22, 1007-1009 (2006).</li><li>de Vries, S. J., van Dijk, M., Bonvin, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+HADDOCK+web+server+for+data-driven+biomolecular+docking.">The HADDOCK web server for data-driven biomolecular docking.</a> Nat. Protoc. 5, 883-897 (2010).</li><li>Capriotti, E., Marti-Renom, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SARA:+a+server+for+function+annotation+of+RNA+structures.">SARA: a server for function annotation of RNA structures.</a> Nucleic Acids Res. 37, 260-265 (2009).</li><li>van Dijk, M., Bonvin, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D-DART:+a+DNA+structure+modelling+server.">3D-DART: a DNA structure modelling server.</a> Nucleic Acids Res. 37, W235-W239 (2009).</li><li>Contreras-Moreira, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D-footprint:+a+database+for+the+structural+analysis+of+protein-DNA+complexes.">3D-footprint: a database for the structural analysis of protein-DNA complexes.</a> Nucleic Acids Res. 38, D91-D97 (2010).</li><li>Popenda, M., Szachniuk, M., Blazewicz, M., Wasik, S., Burke, E. K., Blazewicz, J., Adamiak, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+FRABASE+2.0:+an+advanced+web-accessible+database+with+the+capacity+to+search+the+three-dimensional+fragments+within+RNA+structures.">RNA FRABASE 2.0: an advanced web-accessible database with the capacity to search the three-dimensional fragments within RNA structures.</a> BMC Bioinformatics. 11, 231 (2010).</li><li>&#268;ech, P., Svozil, D., Hoksza, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SETTER:+web+server+for+RNA+structure+comparison.">SETTER: web server for RNA structure comparison.</a> Nucleic Acids Res. , (2012).</li></ol>

Keine Interessenskonflikte erklärt.

Der Satz von Protokollen in diesem Artikel vorgestellt nur von den Fähigkeiten des 3DNA Suite von Programmen zu berühren. Die Werkzeuge angewendet werden, um Strukturen RNA nicht-kanonischen Basenpaaren zu identifizieren, um die sekundären strukturellen Kontexte, in denen eine solche Paarung tritt bestimmen, um die räumliche Anordnung der spiralförmigen Fragmente zu quantifizieren, um die Überlappung von Basen entlang der Kette Rückgrat messen etc. werden Der Umbau Befehl ermöglicht es dem Benutzer, einfach und ...

Das Verständnis der dreidimensionalen Strukturen von DNA, RNA und ihre Komplexe mit Proteinen, Drogen und andere Liganden, ist von entscheidender Bedeutung für die Entschlüsselung ihrer vielfältigen biologischen Funktionen, und zum Ermöglichen das rationale Design von Therapeutika. Exploration solcher Strukturen umfasst drei getrennte, aber eng verwandte Komponenten: Analyse (Muster in Formen und Wechselwirkungen extrahieren), Modellierung (zu Energetik und Molekulardynamik beurteilen) und Visualisierung. Statik und Modellbau sind im Wesentlichen zwei Seiten der gleichen Medaille, und Visualisierung ergänzt sie beide. Die 3DNA Suite von Computerprogrammen ist eine zunehmend beliebte strukturelle Bioinformatik-Toolkit mit Fähigkeiten zu analysieren, zu konstruieren, zu visualisieren und dreidimensionale Strukturen Nukleinsäure. Frühere Veröffentlichungen erläutert die Möglichkeiten der Software 1, sofern Rezepte durchzuführen ausgewählten Aufgaben 2, führte die web-basierte Schnittstellezu beliebten Features der Software 3, gesammelt präsentiert Datenbanken struktureller Merkmale mit 3DNA 4, 5 und veranschaulicht die Nützlichkeit der Software für die Analyse von DNA und RNA-Strukturen 6, 7. Das Ziel dieses Artikels ist es, die 3DNA Software-Kit zur Labor-Wissenschaftler und andere mit Interessen und / oder Bedürfnisse zu bringen, um DNA-und RNA-räumliche Organisation mit state-of-the-art Computer-Tools untersuchen. Die Protokolle hier vorgestellten Schritt-für-Schritt-Anleitung (i) zum Herunterladen und Installieren der Software auf einem Mac OS X-System, (ii-iii) zu analysieren und zu modifizieren DNA-Strukturen auf der Ebene der konstituierenden Basenpaar-Schritten ( iv-v) zu analysieren und auszurichten Gruppen von verwandten DNA-Strukturen, und (vi) die Modelle von Protein-DNA-Ketten dekoriert mit der benutzerfreundlichen Weboberfläche w3DNA konstruieren. Die Software hat die Fähigkeit, zu analysieren einzelnen Strukturen gelöst mit röntgenkristallographische Methoden sowie großeEnsembles von Strukturen mit Kernspinresonanz (NMR) ermittelt oder die durch Computer-Simulation Techniken. Die hier untersuchten Strukturen umfassen (i) die hochauflösende Kristallstruktur von DNA gebunden an die HBB Protein aus Borrelia burgdorferi 8 (die von Zecken übertragene Bakterium, dass Lyme-Borreliose verursacht beim Menschen 9, 10), (ii) zwei große Gruppen von sequentiell verwandten DNA-Moleküle mit Molekülsimulationen 11 erzeugt - 4,500 Momentaufnahmen d (GGCAAAATTTTGCC) 2 und d (CCGTTTTAAAACGG) 2 bei 100-ps-Schritten während der Berechnungen gesammelt, und (iii) eine kleine Ensemble NMR-basierte Strukturen der DNA O3 Bediener verpflichtet, den Kopfschmuck des Escherichia coli Lac Repressorprotein 12. Die folgenden Anweisungen enthalten Informationen darüber, wie die Dateien der Atomkoordinaten mit jeder dieser Strukturen sowie wie 3DNA (Eine Kopie dieser Datei gefunden verwenden assoziiert zugreifenauf der 3DNA Forum auf <a href="http://forum.x3dna.org/jove" target="_blank">http://forum.x3dna.org/jove</a> ) zu prüfen und ändern diese Strukturen.

analyzing and building nucleic acid structures with 3dna

Die 3DNA Softwarepaket ist eine beliebte und vielseitige Bioinformatik-Tool mit Funktionen zu analysieren, zu konstruieren, zu visualisieren und dreidimensionale Strukturen Nukleinsäure. Dieser Artikel stellt ausführliche Protokolle für eine Teilmenge der neue und beliebte Features, die in 3DNA, für beide einzelnen Strukturen und Ensembles von verwandten Strukturen. Protokoll Nr. 1 enthält die Reihe von Anweisungen benötigt zum Herunterladen und Installieren der Software. Darauf folgt in Protokoll 2, durch die Analyse eines Nukleinsäure-Struktur, einschließlich der Zuordnung von Basenpaaren und die Bestimmung der Starrkörper-Parametern, die die Struktur zu beschreiben und in Protokoll Nr. 3, gefolgt von einer Beschreibung der Rekonstruktion eines atomaren Modell einer Struktur aus der Starrkörper-Parameter. Die neueste Version von 3DNA, Version 2.1, verfügt über neue Features für die Analyse und Manipulation von Ensembles von Strukturen, wie jene von Kernspinresonanz (NMR)-Messungen und molekularen Dynamik (MD abgeleitet) Simulationen; diese Funktionen sind in den Protokollen 4 und 5 dargestellt. Neben den Stand-Alone-3DNA Softwarepaket, das w3DNA Webserver an folgenden <a href="http://w3dna.rutgers.edu" target="_blank">http://w3dna.rutgers.edu</a> bietet eine benutzerfreundliche Schnittstelle auf ausgewählte Funktionen der Software. Protokoll Nr. 6 zeigt ein neues Merkmal der Website für den Bau von Modellen der langen DNA-Molekülen mit Proteinen in benutzerspezifischen Orten eingerichtet.

Die 3DNA Software-Tools werden routinemäßig zur Nukleinsäure-Strukturen zu analysieren. Zum Beispiel werden die Identitäten von Basenpaaren und der Starrkörperplatte Parameter, die die Anordnung der Basen in Doppelhelix-DNA-Fragmente und RNA-Strukturen charakterisieren automatisch berechnet und für jeden neuen Eintrag gespeichert in der Nukleinsäure-Datenbank 22, eine weltweite Sammlung von Nukleinsäure strukturelle Informationen. Die Werte der starren Körper Parameter mit Protokoll Nr. 2 bestimmt le...

Watch this Scientific Journal Video about Analyzing and Building Nucleic Acid Structures with 3DNA at JoVE.com

Analyzing and Building Nucleic Acid Structures with 3DNA

Die 3DNA Softwarepaket ist eine beliebte und vielseitige Bioinformatik-Tool mit Funktionen zu analysieren, zu konstruieren, zu visualisieren und dreidimensionale Strukturen Nukleinsäure. Dieser Artikel stellt ausführliche Protokolle für eine Teilmenge der neue und beliebte Features, die in 3DNA, für beide einzelnen Strukturen und Ensembles von verwandten Strukturen.

Analysieren und Bauen Nukleinsäurestrukturen mit 3DNA

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic ...

analyzing-and-building-nucleic-acid-structures-with-3dna

Research

JoVE Journal

Biology

20.4K Aufrufe.  Rutgers - The State University of New Jersey. Die 3DNA Softwarepaket ist eine beliebte und vielseitige Bioinformatik-Tool mit Funktionen zu analysieren, zu konstruieren, zu visualisieren und dreidimensionale Strukturen Nukleinsäure. Dieser Artikel stellt ausführliche Protokolle für eine Teilmenge der neue und beliebte Features, die in 3DNA, für beide einzelnen Strukturen und Ensembles von verwandten Strukturen.

Serie: Analyzing and Building Nucleic Acid Structures with 3DNA

AC Electrokinetic Phenomena Generated by Microelectrode Structures

The field of AC electrokinetics is rapidly growing due to its ability to perform dynamic fluid and particle manipulation on the micro- and nano-scale, which is essential for Lab-on-a-Chip applications. AC electrokinetic phenomena use electric fields to generate forces that act on fluids or suspended particles (including those made of dielectric or biological material) and cause them to move in astonishing ways1, 2. Within a single channel, AC electrokinetics can accomplish many essential on-chip operations such as active micro-mixing, particle separation, particle positioning and micro-pattering. A single device may accomplish several of those operations by simply adjusting operating parameters such as frequency or amplitude of the applied voltage. Suitable electric fields can be readily created by micro-electrodes integrated into microchannels. It is clear from the tremendous growth in this field that AC electrokinetics will likely have a profound effect on healthcare diagnostics3-5, environmental monitoring6 and homeland security7.

In general, there are three AC Electrokinetic phenomena (AC electroosmosis, dielectrophoresis and AC electrothermal effect) each with unique dependencies on the operating parameters. A change in these operating parameters can cause one phenomena to become dominant over another, thus changing the particle or fluid behavior. 

It is difficult to predict the behavior of particles and fluids due to the complicated physics that underlie AC electrokinetics. It is the goal of this publication to explain the physics and elucidate particle and fluid behavior. Our analysis also covers how to fabricate the electrode structures that generate them, and how to interpret a wide number of experimental observations using several popular device designs. This video article will help scientists and engineers understand these phenomena and may encourage them to start using AC Electrokinetics in their research.

Manipulating fluids and suspended particles in the micro- and nano-scale is becoming more of a reality as enabling technologies, like AC electrokinetics, continue to develop. Here, we discuss the physics behind AC electrokinetics, how to fabricate these devices and how to interpret the experimental observations.

AC elektrokinetischen Phänomenen von Mikroelektroden-Strukturen erzeugt

The field of AC electrokinetics is rapidly growing due to its ability to perform dynamic fluid and particle manipulation on the micro- and nano-scale, ...

Forschung

Biologie

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

Färbung von Proteinen in Gelen mit Coomassie G-250 ohne organische Lösungsmittel und Essigsäure

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Fluorescent Labeling of Drosophila Heart Structures

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells that align along each side of the heart wall, supportive alary muscles and, in adults, a layer of ventral longitudinal muscle cells. The contractile fibers house conserved constituents of the muscle cytoarchitecture including densely packed bundles of myofibrils and cytoskeletal/submembranous protein complexes, which interact with homologous components of the extracellular matrix. Here we describe a protocol for the fixation and the fluorescent labeling of particular myocardial elements from the hearts of dissected larvae and semi-intact adult Drosophila. Specifically, we demonstrate the labeling of sarcomeric F-actin and of &alpha;-actinin in larval hearts. Additionally, we perform labeling of F-actin and &alpha;-actinin in myosin-GFP expressing adult flies and of &alpha;-actinin and pericardin, a type IV extracellular matrix collagen, in wild type adult hearts. Particular attention is given to a mounting strategy for semi-intact adult hearts that minimizes handling and optimizes the opportunity for maintaining the integrity of the cardiac tubes and the associated tissues. These preparations are suitable for imaging via fluorescent and confocal microscopy. Overall, this procedure allows for careful and detailed analysis of the structural characteristics of the heart from a powerful genetically tractable model system.

Fluorescent Labeling of Drosophila Heart Structures

Here we describe a basic protocol for fluorescent labeling of different elements of heart tubes from larva and adult Drosophila melanogaster. These specimens are well-suited for imaging via fluorescent or confocal microscopy. This technique permits detailed structural analysis of the features of the hearts from a powerful model organism.

Fluoreszenzmarkierung Drosophila Herz Structures

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells ...

Electricity-Free, Sequential Nucleic Acid and Protein Isolation

Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern for governments, industries and medical professionals worldwide. For example, EHECs, combined with Shigella, are responsible for the deaths of approximately 325,000 children each year and are particularly prevalent in the developing world where laboratory-based identification, common in the United States, is unavailable 1. The development and distribution of low cost, field-based, point-of-care tools to aid in the rapid identification and/or diagnosis of pathogens or disease markers could dramatically alter disease progression and patient prognosis. We have developed a tool to isolate nucleic acids and proteins from a sample by solid-phase extraction (SPE) without electricity or associated laboratory equipment 2. The isolated macromolecules can be used for diagnosis either in a forward lab or using field-based point-of-care platforms. Importantly, this method provides for the direct comparison of nucleic acid and protein data from an un-split sample, offering a confidence through corroboration of genomic and proteomic analysis.
Our isolation tool utilizes the industry standard for solid-phase nucleic acid isolation, the BOOM technology, which isolates nucleic acids from a chaotropic salt solution, usually guanidine isothiocyanate, through binding to silica-based particles or filters 3. CUBRC's proprietary solid-phase extraction chemistry is used to purify protein from chaotropic salt solutions, in this case, from the waste or flow-thru following nucleic acid isolation4.
By packaging well-characterized chemistries into a small, inexpensive and simple platform, we have generated a portable system for nucleic acid and protein extraction that can be performed under a variety of conditions. The isolated nucleic acids are stable and can be transported to a position where power is available for PCR amplification while the protein content can immediately be analyzed by hand held or other immunological-based assays. The rapid identification of disease markers in the field could significantly alter the patient's outcome by directing the proper course of treatment at an earlier stage of disease progression. The tool and method described are suitable for use with virtually any infectious agent and offer the user the redundancy of multi-macromolecule type analyses while simultaneously reducing their logistical burden.

A tool and chemistries are described to sequentially isolate nucleic acids followed by proteins from a sample without the need for electricity. The tool consists of a sorbent held within a transfer pipette while the isolation chemistries are based on solid-phase extraction principles. The isolated macromolecules can be analyzed by immuno-based and PCR-based assays.

Strom-Free, Sequential Nukleinsäure-und Protein Isolation

Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern ...

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or&#160;photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.

We demonstrate the use of fluorescence photo activation localization microscopy (FPALM) to simultaneously image multiple types of fluorescently labeled molecules&#160;within cells. The techniques described yield the localization of thousands to hundreds of thousands of individual fluorescent labeled proteins, with a precision of tens of nanometers&#160;within single cells.

Simultane Multicolor-Imaging biologischer Strukturen mit Fluoreszenzphotoaktivierung Lokalisationsmikroskopie

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled ...

The Fastest Western in Town: A Contemporary Twist on the Classic Western Blot Analysis

The Western blot techniques that were originally established in the late 1970s are still actively utilized today. However, this traditional method of Western blotting has several drawbacks that include low quality resolution, spurious bands, decreased sensitivity, and poor protein integrity. Recent advances have drastically improved numerous aspects of the standard Western blot protocol to produce higher qualitative and quantitative data. The Bis-Tris gel system, an alternative to the conventional Laemmli system, generates better protein separation and resolution, maintains protein integrity, and reduces electrophoresis to a 35 min run time. Moreover, the iBlot dry blotting system, dramatically improves the efficacy and speed of protein transfer to the membrane in 7 min, which is in contrast to the traditional protein transfer methods that are often more inefficient with lengthy transfer times. In combination with these highly innovative modifications, protein detection using infrared fluorescent imaging results in higher-quality, more accurate and consistent data compared to the standard Western blotting technique of chemiluminescence. This technology can simultaneously detect two different antigens on the same membrane by utilizing two-color near-infrared dyes that are visualized in different fluorescent channels. Furthermore, the linearity and broad dynamic range of fluorescent imaging allows for the precise quantification of both strong and weak protein bands. Thus, this protocol describes the key improvements to the classic Western blotting method, in which these advancements significantly increase the quality of data while greatly reducing the performance time of this experiment.

This protocol explores the latest advancements in performing Western blot analyses.&#160;These novel modifications employ a Bis-Tris gel system with a 35&#160;min electrophoresis run time, a 7&#160;min dry blotting transfer system, and infrared fluorescent protein detection and imaging that generates higher resolution, quality, sensitivity, and improved accuracy of Western data.

Der schnellste westlichen Stadt: Eine moderne Variante der Classic Western-Blot-Analyse

The Western blot techniques that were originally established in the late 1970s are still actively utilized today. However, this traditional method of ...

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5&#8217;-ATCGAT-3&#8217; sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for methyltransferase-directed Transfer of Activated Groups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as well as DNA genotyping and methylation detection.

DNA and proteins are sequence-specifically labeled with affinity or fluorescent reporter groups using DNA or protein methyltransferases and synthetic cofactor analogues. Depending on the cofactor specificity of the enzymes, aziridine or double activated cofactor analogues are employed for one- or two-step labeling.

Sequenzspezifische Markierung von Nukleinsäuren und Proteinen mit Methyltransferasen und Cofaktor-Analoga

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific ...

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are still not routinely applied, due to the cost and specific equipment associated with commercially available methods. Here we present a simplified method for readout of digital droplet assays using a conventional real-time PCR instrument to measure bulk fluorescence of droplet-based digital assays.
We characterize the performance of the bulk readout assay using synthetic droplet mixtures and a droplet digital multiple displacement amplification (MDA) assay. Quantitative MDA particularly benefits from a digital reaction format, but our new method&#160;applies to any digital assay. For established digital assay protocols such as digital PCR, this method serves to speed up and simplify assay readout.
Our bulk readout methodology brings the advantages of partitioned assays without the need for specialized readout instrumentation. The principal limitations of the bulk readout methodology are reduced dynamic range compared with droplet-counting platforms and the need for a standard sample, although the requirements for this standard are less demanding than for a conventional real-time experiment. Quantitative whole genome amplification (WGA) is used to test for contaminants in WGA reactions and is the most sensitive way to detect the presence of DNA fragments with unknown sequences, giving the method great promise in diverse application areas including pharmaceutical quality control and astrobiology.

We describe an endpoint digital assay for quantifying nucleic acids with a simplified (analog) readout. We measure bulk fluorescence of droplet-based digital assays using a standard qPCR machine rather than specialized instrumentation and confirm our results by microscopy.

Einfache Massen Auslesen des digitalen Nukleinsäure Quantifizierung Assays

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are ...

Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing technologies have allowed for the rapid and efficient characterization of bacterial communities using 16S rRNA gene sequencing from a variety of sources. Although readily available tools for 16S rRNA sequence analysis have standardized computational workflows, sample processing for DNA extraction remains a continued source of variability across studies. Here we describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs. We also delineate downstream methods for 16S rRNA gene sequencing, including generation of sequencing libraries, data quality control, and sequence analysis. The workflow can accommodate multiple samples types, including stool and swabs collected from a variety of anatomical locations and host species. Additionally, recovered DNA and RNA can be separated and used for other applications, including whole genome sequencing or RNA-seq. The method described allows for a common processing approach for multiple sample types and accommodates downstream analysis of genomic, metagenomic and transcriptional information.

We describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs for characterization of bacterial communities using 16S rRNA gene amplicon sequencing. The method allows for a common processing approach for multiple sample types and accommodates a number of downstream analytic processes.

Effiziente Nukleinsäure-Extraktion und 16S rRNA Gen-Sequenzierung für bakterielle Gemeinschaft Charakterisierung

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing ...

Laser-Capture Microdissection RNA-Sequencing for Spatial and Temporal Tissue-Specific Gene Expression Analysis in Plants

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify transcriptional regulatory networks that govern developmental processes it is necessary to measure the spatial and temporal gene expression profiles of these individual cell types. Therefore, insight into the spatio-temporal control of gene expression is essential to gain understanding of how biological and developmental processes are regulated. Here, we describe a laser-capture microdissection (LCM) method to isolate small number of cells from three barley embryo organs over a time-course during germination followed by transcript profiling. The method consists of tissue fixation, tissue processing, paraffin embedding, sectioning, LCM and RNA extraction followed by real-time PCR or RNA-seq. This method has enabled us to obtain spatial and temporal profiles of seed organ transcriptomes from varying numbers of cells (tens to hundreds), providing much greater tissue-specificity than typical bulk-tissue analyses. From these data we were able to define and compare transcriptional regulatory networks as well as predict candidate regulatory transcription factors for individual tissues. The method should be applicable to other plant tissues with minimal optimization.

Presented here is a protocol for laser-capture microdissection (LCM) of plant tissues. LCM is a microscopic technique for isolating areas of tissue in a contamination-free manner. The procedure includes tissue fixation, paraffin embedding, sectioning, LCM and RNA extraction. RNA is used in the downstream tissue-specific, temporally resolved analysis of transcriptomes.