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

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 ...

Frederick National Laboratory for Cancer Research

A multiple antigen peptide display scaffold was used to create multivalent versions of a heptapeptide selected previously by phage display to bind to Bacillus subtilis spores. A simple flow cytometric assay was developed in which a biotinylated form of the peptide was first bound to fluorescent streptavidin, then the fluorescent streptavidin-peptide complex was bound to spores before introduction into the cytometer. This assay clearly demonstrated that the tetravalent scaffold enhanced the affinity for B. subtilis spores by greater than 1 and 2 orders of magnitude when compared to divalent and monovalent analogues, respectively. However, variations in the number and flexibility of spacer residues within the scaffold did not significantly affect the binding affinity of the tetravalent peptides. Similar to prior reports, these multivalent scaffolds are effective most likely because they mimic the multivalent display of the original peptide library on the phage coat. Moreover, the tetravalent peptides can be readily integrated into a variety of heterogeneous and homogeneous spore-detection assay formats.

Refined multivalent display of bacterial spore-binding peptides.

Un &eacute;chafaudage d&#39;affichage de peptide antig&egrave;ne multiples a &eacute;t&eacute; utilis&eacute; pour cr&eacute;er des versions d&#39;un heptapeptide s&eacute;lectionn&eacute; pr&eacute;c&eacute;demment par le phage display pour lier aux spores de Bacillus subtilis MULTIVALENTES. Un simple flux en cytom&eacute;trie de flux a &eacute;t&eacute; d&eacute;velopp&eacute; dans lequel une forme biotinyl&eacute; du peptide devait d&#39;abord streptavidine fluorescente, puis le complexe streptavidine-peptide fluorescent a &eacute;t&eacute; li&eacute; &agrave; spores avant leur introduction dans le cytom&egrave;tre. Ce dosage a clairement d&eacute;montr&eacute; que l&#39;&eacute;chafaud t&eacute;travalent renforc&eacute;e l&#39;affinit&eacute; pour les spores de b. subtilis par plus de 1 &agrave; 2 ordres de grandeur par rapport aux analogues divalents et monovalents, respectivement. Toutefois, les variations du nombre et de la flexibilit&eacute; des r&eacute;sidus d&#39;entretoise au sein de l&#39;&eacute;chafaud n&#39;affectaient pas significativement l&#39;affinit&eacute; des peptides t&eacute;travalents. Similaire &agrave; pr&eacute;c&eacute;dents rapports, ces &eacute;chafaudages multivalents sont efficaces tr&egrave;s probablement parce qu&#39;ils imitent l&#39;affichage multivalent de la biblioth&egrave;que de peptide original sur le manteau du bact&eacute;riophage. En outre, les peptides t&eacute;travalents peuvent &ecirc;tre facilement int&eacute;gr&eacute;s dans une vari&eacute;t&eacute; de formats de dosage de d&eacute;tection des spores h&eacute;t&eacute;rog&egrave;nes et homog&egrave;nes.

Affichage de multivalent raffin&eacute; des peptides de liaison de spores bact&eacute;riennes.

Un multiple antigene peptidico display ponteggio &egrave; stato utilizzato per creare versioni polivalente di un eptapeptide selezionato in precedenza dall&#39;esposizione dei fagi per associare a spore di Bacillus subtilis. Un dosaggio di cytometric di flusso semplice &egrave; stato sviluppato in cui una forma di anticorpo del peptide prima era associata a streptavidina fluorescente, poi il complesso fluorescente streptavidina-peptide &egrave; stato associato a spore prima introduzione il citometro. Questa analisi ha chiaramente dimostrato che il patibolo tetravalente rafforzata l&#39;affinit&agrave; per le spore di b. subtilis da maggiore di 1 e 2 ordini di grandezza rispetto agli analoghi monovalente e bivalente, rispettivamente. Tuttavia, variazioni del numero e la flessibilit&agrave; dei residui distanziale entro il patibolo non hanno influito in modo significativo l&#39;affinit&agrave; di legame dei peptidi tetravalente. Simile ai precedenti rapporti, questi ponteggi polivalente sono efficaci molto probabilmente perch&eacute; essi imitano il display polivalente della biblioteca del peptide originale sul cappotto dei fagi. Inoltre, i peptidi tetravalente possono essere facilmente integrati in una variet&agrave; di formati di dosaggio eterogeneo e omogeneo spora-rilevazione.

Raffinato display polivalente di peptidi associazione spore batteriche.

The synthesis and full 3D structural characterization of nine new 1-benzenesulfonyl-2-methyl-1,2,3,4-tetrahydroquinoline derivatives are reported. These belong to a library whose rationale for the design was the previous knowledge of the biological relevant properties of both structural moieties. From protozoan antiparasitic screening, compounds 3 demonstrated interesting activity against Trypanozoma cruzi with low cytotoxicity. Besides, most compounds were moderately active against Plasmodium falciparum. Of these, 3 and 9 can be considered as lead scaffolds for further optimization. The substituent on BS did not influence the 3D structure properties and the (1)H NMR spectra revealed the existence of an intramolecular weak hydrogen bond, C-Hcdots, three dots, centeredOS. Molecular modeling and X-ray crystallography also confirmed this finding, which is relevant to compound conformational preference.

Synthesis, stereoelectronic characterization and antiparasitic activity of new 1-benzenesulfonyl-2-methyl-1,2,3,4-tetrahydroquinolines.

La synth&egrave;se et la caract&eacute;risation compl&egrave;te en 3D de neuf nouveaux d&eacute;riv&eacute;s 1-benzenesulfonyl-2-methyl-1,2,3,4-tetrahydroquinoline sont rapport&eacute;s. Ceux-ci appartiennent &agrave; une biblioth&egrave;que dont la rationalit&eacute; de la conception a &eacute;t&eacute; la connaissance pr&eacute;alable des propri&eacute;t&eacute;s biologiques pertinentes des deux moiti&eacute;s structurelles. De d&eacute;pistage antiparasitaire protozoaire, compos&eacute;s 3 a d&eacute;montr&eacute; une activit&eacute; int&eacute;ressante Trypanozoma cruzi avec faible cytotoxicit&eacute;. En outre, la plupart compos&eacute;s &eacute;taient mod&eacute;r&eacute;ment actifs contre Plasmodium falciparum. Parmi eux, 3 et 9 peut &ecirc;tre consid&eacute;r&eacute; comme &eacute;chafaudages de plomb pour l&#39;optimisation suppl&eacute;mentaire. Le substituant sur BS n&#39;influence pas les propri&eacute;t&eacute;s de la structure 3D et les spectres de RMN de H (1) a r&eacute;v&eacute;l&eacute; l&#39;existence d&#39;une liaison hydrog&egrave;ne intramol&eacute;culaire faible, C-Hcdots, trois points, centeredOS. Mod&eacute;lisation mol&eacute;culaire et la cristallographie aux rayons x a &eacute;galement confirment cette conclusion, qui se rapporte &agrave; une pr&eacute;f&eacute;rence conformationnelle compos&eacute;e.

Synth&egrave;se, caract&eacute;risation st&eacute;r&eacute;o&eacute;lectroniques et activit&eacute; antiparasitaire du nouveau 1-benzenesulfonyl-2-methyl-1,2,3,4-tetrahydroquinolines.

Sintesi e caratterizzazione strutturale 3D completa di nove nuovi derivati 1-benzenesulfonyl-2-methyl-1,2,3,4-tetrahydroquinoline sono segnalati. Questi appartengono a una libreria di cui razionale per la progettazione &egrave; stata la precedente conoscenza delle propriet&agrave; biologiche rilevanti di entrambi moiety strutturali. Da protozoo screening antiparassitari, composti 3 ha dimostrato attivit&agrave; interessante contro Trypanozoma cruzi con bassa citotossicit&agrave;. Inoltre, la maggior parte dei composti erano moderatamente attivi contro il Plasmodium falciparum. Di questi, 3 e 9 pu&ograve; essere considerato come piombo ponteggi per ulteriore ottimizzazione. Il sostituente su BS non influenza le propriet&agrave; della struttura 3D e gli spettri NMR di H (1) ha rivelato l&#39;esistenza di un legame idrogeno debole intramolecolare, C-Hcdots, tre puntini, centeredOS. Modellazione molecolare e cristallografia a raggi x ha anche confermato questa constatazione, che &egrave; rilevante per i composto preferenza conformazionale.

Sintesi, caratterizzazione stereoelectronic e antiparassitari attivit&agrave; di nuovo 1-benzenesulfonyl-2-methyl-1,2,3,4-tetrahydroquinolines.

Targeting guanine (G) quadruplex structures is an exciting new strategy with potential for controlling gene expression and designing anticancer agents. Guanine-rich peptide nucleic acid (PNA) oligomers bind to homologous DNA and RNA to form hetero-G-quadruplexes but can also bind to complementary cytosine-rich sequences to form heteroduplexes. In this study, we incorporated backbone modifications into G-rich PNAs to improve the selectivity for quadruplex versus duplex formation. Incorporation of abasic sites as well as chiral modifications to the backbone were found to be effective strategies for improving selectivity as shown by UV-melting and surface plasmon resonance measurements. The enhanced selectivity is due primarily to decreased affinity for complementary sequences, since binding to the homologous DNA to form PNA-DNA heteroquadruplexes retains high affinity. The improved selectivity of these PNAs is an important step toward using PNAs for regulating gene expression by G-quadruplex formation.

Loop and backbone modifications of peptide nucleic acid improve g-quadruplex binding selectivity.

Ciblage quadruplex de guanine (G) des structures est une nouvelle strat&eacute;gie passionnante avec potentiel pour contr&ocirc;ler l&#39;expression des g&egrave;nes et de concevoir des agents anticanc&eacute;reux. Oligom&egrave;res d&#39;acides nucl&eacute;iques (PNA) de peptide riche en guanine lient &agrave; l&#39;ADN et l&#39;ARN homologue &agrave; forme h&eacute;t&eacute;ro-G-quadruplexes mais peuvent &eacute;galement lier &agrave; des s&eacute;quences riches en cytosine compl&eacute;mentaires &agrave; forme h&eacute;t&eacute;roduplex. Dans cette &eacute;tude, nous avons incorpor&eacute; modification de la colonne vert&eacute;brale dans PNAs riches en G pour am&eacute;liorer la s&eacute;lectivit&eacute; pour quadruplex contre formation recto verso. Incorporation des sites abasiques modifications ainsi que chirale &agrave; la colonne vert&eacute;brale se sont r&eacute;v&eacute;l&eacute;es des strat&eacute;gies efficaces pour am&eacute;liorer la s&eacute;lectivit&eacute; comme indiqu&eacute; par la fonte des UV et des mesures de r&eacute;sonance plasmon de surface. La s&eacute;lectivit&eacute; accrue s&#39;explique principalement par une diminution d&#39;affinit&eacute; pour les s&eacute;quences compl&eacute;mentaires, &eacute;tant donn&eacute; que la liaison &agrave; l&#39;ADN homologue pour former heteroquadruplexes PNA-ADN conserve haute affinit&eacute;. La s&eacute;lectivit&eacute; am&eacute;lior&eacute;e de ces PNAs est une &eacute;tape importante vers l&#39;utilisation de PNAs de r&eacute;gulation de l&#39;expression g&eacute;nique par G-quadruplexe formation.

Modifications de boucle et de la colonne vert&eacute;brale de l&#39;acide nucl&eacute;ique peptidique am&eacute;liorent la s&eacute;lectivit&eacute; de liaison g-quadruplexe.

Targeting quadruplex guanina (G) strutture &egrave; un&#39;eccitante nuova strategia con potenziale per controllare l&#39;espressione genica e la progettazione di agenti antitumorali. Oligomeri di acidi nucleici (PNA) del peptide ricco di guanina associare all&#39;omologo del DNA e RNA a forma etero-G-quadruplex ma possono anche associare a complementare sequenze citosina-ricco di forma heteroduplexes. In questo studio, abbiamo incorporato le modifiche backbone in PNAs G-ricchi per migliorare la selettivit&agrave; per quadruplex versus formazione duplex. Incorporazione di abasic siti cos&igrave; come chirali modifiche alla colonna vertebrale sono state trovate per essere efficaci strategie per migliorare la selettivit&agrave; come mostrato dalla fusione di raggi UV e le misure di risonanza plasmonica di superficie. La maggiore selettivit&agrave; &egrave; dovuta principalmente alla ridotta affinit&agrave; per le sequenze complementari, dal momento che mantiene il legame al DNA omologo per formare heteroquadruplexes PNA-DNA ad alta affinit&agrave;. La migliore selettivit&agrave; di questi PNAs &egrave; un passo importante verso usando PNAs per la regolazione della espressione genica di formazione di G-quadruplex.

Modifiche di loop e spina dorsale dell&#39;acido peptidonucleico migliorano la selettivit&agrave; di associazione di g-quadruplex.

Ty1&#xFF0C;&#x917F;&#x9152;&#xFF0C;&#x957F;&#x7EC8;&#x7AEF;&#x91CD;&#x590D;&#x5EA7;&#x5B50;&#x7ED3;&#x6784;&#x4E0A;&#x548C;&#x529F;&#x80FD;&#x4E0A;&#x76F8;&#x5173;&#x7684;&#x9006;&#x8F6C;&#x5F55;&#x75C5;&#x6BD2;&#x3002;&#x7136;&#x800C;&#xFF0C;&#x8FD9;&#x4E9B; retroelements &#x4E4B;&#x95F4;&#x7684;&#x533A;&#x5206;&#x65B9;&#x9762;&#x662F;&#x4F7F;&#x7528;&#x957F;&#x7EC8;&#x7AEF;&#x91CD;&#x590D;&#x53CD;&#x8F6C;&#x5F55;&#x8F6C;&#x5EA7;&#x5B50;&#x7684;&#x590D;&#x5236;&#x7B56;&#x7565;&#x7684;&#x591A;&#x6837;&#x6027;&#x3002;&#x82E5;&#x8981;&#x4E86;&#x89E3;&#x72EC;&#x8054;&#x4F53;&#x4EE3;&#x7406; GAG &#x533A;&#x57DF;&#x4ECE;&#x672C; Ty1 &#x57FA;&#x56E0;&#x7EC4; rna &#x63A7;&#x5236;&#x5143;&#x7D20;&#x53CD;&#x5411;&#x8F6C;&#x5F55;&#x7684;&#x7ED3;&#x6784;&#x7EC4;&#x7EC7;&#xFF0C;&#x5E94;&#x7528;&#x5316;&#x5B66;&#x9176;&#x63A2;&#x8BA8;&#x5411; RNA/tRNA &#x590D;&#x5408;&#x7269;&#x4F53;&#x5916;&#x7EC4;&#x88C5;&#x548C; RNA &#x75C5;&#x6BD2;&#x6837;&#x9897;&#x7C92;&#x3002;&#x901A;&#x8FC7;&#x6BD4;&#x8F83;&#x4E0D;&#x540C;&#x7684; RNA &#x56FD;&#x5BB6;&#xFF0C;&#x6211;&#x4EEC;&#x7684;&#x5206;&#x6790;&#x63D0;&#x4F9B;&#x5168;&#x9762;&#x7684;&#x7ED3;&#x6784;&#x7684;&#x5E95;&#x6F06;&#x7ED1;&#x5B9A;&#x7AD9;&#x70B9;&#x3001; &#x6BD7;&#x90BB;&#x5F15;&#x7269;&#x7ED3;&#x5408;&#x4F4D;&#x70B9;&#x7684;&#x65B0;&#x578B; pseudoknot&#x3001; &#x542B;&#x590D;&#x53D1;&#x6027;&#x5E8F;&#x5217; RNA &#x4E8C;&#x805A;&#x6216;&#x5305;&#x88C5;&#x548C;&#x5019;&#x9009;&#x86CB;&#x767D;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x7AD9;&#x70B9;&#x4E2D;&#x53EF;&#x80FD;&#x6D89;&#x53CA;&#x7684;&#x4E09;&#x4E2A;&#x533A;&#x57DF;&#x3002;&#x6B64;&#x5916;&#xFF0C;&#x6211;&#x4EEC;&#x786E;&#x5B9A;&#x7684;&#x8F6C;&#x5EA7;&#x5B50;&#x63A7;&#x5236;&#x57FA;&#x4E8E;&#x5173;&#x8054;&#x75C5;&#x6BD2;&#x6837;&#x9897;&#x7C92;&#x7684; Ty1 &#x53CD;&#x4E49;&#x8A8A;&#x672C;&#x5C0F;&#x8BF4;&#x7684;&#x5F62;&#x5F0F;&#x7684;&#x5F71;&#x54CD;&#x3002;&#x6211;&#x4EEC;&#x7684;&#x7814;&#x7A76;&#x7ED3;&#x679C;&#x652F;&#x6301;&#x53CD;&#x4E49; Rna &#x6291;&#x5236; retrotransposition&#xFF0C;&#x901A;&#x8FC7;&#x9488;&#x5BF9; Ty1 &#x86CB;&#x767D;&#x8D28;&#x529F;&#x80FD;&#xFF0C;&#x800C;&#x4E0D;&#x662F;&#x9000;&#x706B;&#x7684; RNA &#x57FA;&#x56E0;&#x7EC4;&#x7684;&#x60F3;&#x6CD5;&#x3002;

&#x63A2;&#x7D22; Ty1 &#x53CD;&#x8F6C;&#x5F55;&#x8F6C;&#x5EA7;&#x5B50; RNA &#x7ED3;&#x6784;&#x5185;&#x75C5;&#x6BD2;&#x6837;&#x9897;&#x7C92;&#x3002;

Ty1, a long terminal repeat retrotransposon of Saccharomyces, is structurally and functionally related to retroviruses. However, a differentiating aspect between these retroelements is the diversity of the replication strategies used by long terminal repeat retrotransposons. To understand the structural organization of cis-acting elements present on Ty1 genomic RNA from the GAG region that control reverse transcription, we applied chemoenzymatic probing to RNA/tRNA complexes assembled in vitro and to the RNA in virus-like particles. By comparing different RNA states, our analyses provide a comprehensive structure of the primer-binding site, a novel pseudoknot adjacent to the primer-binding sites, three regions containing palindromic sequences that may be involved in RNA dimerization or packaging and candidate protein interaction sites. In addition, we determined the impact of a novel form of transposon control based on Ty1 antisense transcripts that associate with virus-like particles. Our results support the idea that antisense RNAs inhibit retrotransposition by targeting Ty1 protein function rather than annealing with the RNA genome.

Exploring Ty1 retrotransposon RNA structure within virus-like particles.

Ty1, un long terminal r&eacute;trotransposon r&eacute;p&eacute;ter des Saccharomyces, est structurellement et fonctionnellement li&eacute;es aux r&eacute;trovirus. Cependant, un aspect de diff&eacute;renciation entre ces r&eacute;tro&eacute;l&eacute;ments est la diversit&eacute; des strat&eacute;gies de r&eacute;plication utilis&eacute; par les r&eacute;trotransposons r&eacute;p&eacute;ter long terminales. Pour comprendre l&#39;organisation structurale des intramol&eacute;culaires &eacute;l&eacute;ments pr&eacute;sents sur Ty1 ARN g&eacute;nomique de la r&eacute;gion GAG qui contr&ocirc;lent la transcriptase inverse, nous avons appliqu&eacute; chimioenzymatique sonder de RNA/ARNt complexes assembl&eacute;s in vitro et de l&#39;ARN dans les Pseudo-particules virales. En comparant les diff&eacute;rents &Eacute;tats de RNA, nos analyses fournissent une structure globale de l&#39;appr&ecirc;t-accepteur, un roman pseudon&oelig;ud adjacent aux sites Appr&ecirc;t liant, trois r&eacute;gions contenant des s&eacute;quences palindromiques qui peuvent &ecirc;tre impliqu&eacute;s dans les sites d&#39;interaction prot&eacute;ine dim&eacute;risation ou emballage et candidat RNA. En outre, nous avons d&eacute;termin&eacute; l&#39;impact d&#39;une nouvelle forme de contr&ocirc;le de transposon issu des transcriptions Ty1 qui associent des Pseudo-particules virales. Nos r&eacute;sultats soutiennent l&#39;id&eacute;e que les ARN antisens inhibe r&eacute;trotransposition en ciblant la fonction des prot&eacute;ines Ty1 plut&ocirc;t que de recuit avec l&#39;ARN g&eacute;nomique.

Explorer la structure d&#39;ARN de r&eacute;trotransposons Ty1 dans Pseudo-particules virales.

Ty1, eine lange terminal wiederholen Retrotransposon von Saccharomyces, ist strukturell und funktionell mit Retroviren verwandt. Ein ausschlaggebenden Aspekt zwischen diesen Retroelements ist jedoch die Vielfalt der Replikationsstrategien von long terminal repeat Retrotransposons verwendet. Zum Verst&auml;ndnis der strukturellen Organisation der Cis wirkende Elemente auf Ty1 genomische RNA aus der GAG-Region vorhanden, die reverse Transkription Steuern angewendet wir Chemoenzymatic zu sondieren, um RNA/tRNA-komplexe, die in Vitro zusammengebaut und die RNA in Virus-&auml;hnliche Partikel. Durch den Vergleich verschiedener RNA-betreffen, bieten unsere Analysen eine umfassende Struktur die Z&uuml;ndkapsel-Bindungsstelle, eine neuartige Pseudoknoten angrenzend an die Grundierung-Bindungsstellen, drei Regionen mit palindromische Sequenzen, die in RNA Dimerisierung oder Verpackung und Kandidat Protein Interaktion Websites beteiligt sein k&ouml;nnen. Dar&uuml;ber hinaus haben wir die Auswirkungen der eine neue Form von Transposon-Steuerelement basierend auf Ty1 antisense Abschriften, die Virus-&auml;hnliche Partikel zuordnen festgestellt. Unsere Ergebnisse unterst&uuml;tzen die Idee, dass antisense RNAs Retrotransposition hemmen, indem Sie gezielt Ty1 Proteinfunktion, anstatt Sie zu gl&uuml;hen mit das RNA-Genom.

Erkunden Ty1 Retrotransposon RNA Struktur innerhalb virus&auml;hnliche Partikel.

Ty1, un lungo terminale retrotrasposone ripetizione di Saccharomyces, &egrave; strutturalmente e funzionalmente correlati a retrovirus. Tuttavia, un aspetto di differenziazione tra queste retroelements &egrave; la diversit&agrave; delle strategie replica utilizzato da retrotrasposoni ripetizione terminale lunga. Per comprendere l&#39;organizzazione strutturale degli elementi cis-acting presenti sul RNA genomico Ty1 dalla regione di GAG che controllano la trascrizione inversa, abbiamo applicato chemoenzymatic sondaggio complessi RNA/tRNA assemblati in vitro e il RNA in particelle virus-simili. Confrontando i diversi stati di RNA, le nostre analisi forniscono una struttura completa del sito di legame dell&#39;iniettore, un romanzo pseudoknot adiacente per i siti di legame dell&#39;iniettore, tre regioni contenenti sequenze palindromi che possono essere coinvolti in RNA candidati o imballaggio e dimerizzazione della proteina interazione siti. Inoltre, abbiamo determinato l&#39;impatto di una nuova forma di controllo trasposone basato sulle trascrizioni Ty1 che associano con particelle virus-simili. I nostri risultati supportano l&#39;idea che RNA antisenso inibisce la retrotrasposizione di targeting Ty1 della funzione delle proteine piuttosto che di ricottura con il genoma a RNA.

Esplorare la struttura di RNA di retrotrasposoni Ty1 all&#39;interno particelle virus-simili.

&#x30EC;&#x30C8;&#x30ED;&#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30DD;&#x30BE;&#x30F3; Ty1&#x3001;&#x9577;&#x3044;&#x30BF;&#x30FC;&#x30DF;&#x30CA;&#x30EB; &#x30EA;&#x30D4;&#x30FC;&#x30C8; &#x30EC;&#x30C8;&#x30ED;&#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30DD;&#x30BE;&#x30F3;&#x306E;&#x51FA;&#x82BD;&#x9175;&#x6BCD;&#x3001;&#x69CB;&#x9020;&#x7684;&#x3001;&#x6A5F;&#x80FD;&#x7684;&#x30EC;&#x30C8;&#x30ED; &#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x306B;&#x95A2;&#x9023;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x305F;&#x3060;&#x3057;&#x3001;&#x3053;&#x308C;&#x3089;&#x306E; retroelements &#x306E;&#x9593;&#x306E;&#x5DEE;&#x5225;&#x5316;&#x5074;&#x9762;&#x9577;&#x3044;&#x30BF;&#x30FC;&#x30DF;&#x30CA;&#x30EB;&#x306E;&#x30EA;&#x30D4;&#x30FC;&#x30C8; &#x30EC;&#x30C8;&#x30ED;&#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30DD;&#x30BE;&#x30F3;&#x306B;&#x3088;&#x3063;&#x3066;&#x4F7F;&#x7528;&#x3055;&#x308C;&#x308B;&#x30EC;&#x30D7;&#x30EA;&#x30B1;&#x30FC;&#x30B7;&#x30E7;&#x30F3;&#x6226;&#x7565;&#x306E;&#x591A;&#x69D8;&#x6027;&#x3067;&#x3059;&#x3002;&#x9006;&#x306E;&#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30AF;&#x30EA;&#x30D7;&#x30B7;&#x30E7;&#x30F3;&#x3092;&#x5236;&#x5FA1;&#x3059;&#x308B; cis &#x6F14;&#x6280;&#x8981;&#x7D20;&#x30AE;&#x30E3;&#x30B0;&#x9818;&#x57DF;&#x304B;&#x3089;&#x30EC;&#x30C8;&#x30ED;&#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30DD;&#x30BE;&#x30F3; Ty1 &#x30B2;&#x30CE;&#x30E0; RNA &#x306B;&#x5B58;&#x5728;&#x306E;&#x69CB;&#x9020;&#x7D44;&#x7E54;&#x3092;&#x7406;&#x89E3;&#x3059;&#x308B;&#x306B;&#x306F;&#x3001;&#x5316;&#x5B66;&#x3092; in vitro &#x3067;&#x7D44;&#x307F;&#x7ACB;&#x3066; RNA/tRNA &#x8907;&#x5408;&#x4F53;&#x304A;&#x3088;&#x3073; RNA &#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x69D8;&#x7C92;&#x5B50;&#x306E;&#x30D7;&#x30ED;&#x30FC;&#x30D3;&#x30F3;&#x30B0;&#x3092;&#x9069;&#x7528;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;RNA &#x306E;&#x7570;&#x306A;&#x308B;&#x72B6;&#x614B;&#x3092;&#x6BD4;&#x8F03;&#x3059;&#x308B;&#x3053;&#x3068;&#x306B;&#x3088;&#x3063;&#x3066;&#x3001;&#x79C1;&#x305F;&#x3061;&#x306E;&#x5206;&#x6790;&#x306F;&#x30D7;&#x30E9;&#x30A4;&#x30DE;&#x30FC;&#x7D50;&#x5408;&#x30B5;&#x30A4;&#x30C8;&#x3001;&#x5C0F;&#x8AAC;&#x30B7;&#x30E5;&#x30FC;&#x30C9; &#x30CE;&#x30C3;&#x30C8;&#x306E;&#x30D7;&#x30E9;&#x30A4;&#x30DE;&#x30FC;&#x7D50;&#x5408;&#x90E8;&#x4F4D;&#x306B;&#x96A3;&#x63A5;&#x3059;&#x308B;&#x3001;RNA &#x306E;&#x30C0;&#x30A4;&#x30DE;&#x30FC;&#x5316;&#x307E;&#x305F;&#x306F;&#x5305;&#x88C5;&#x3068;&#x5019;&#x88DC;&#x8005;&#x306E;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x306E;&#x30B5;&#x30A4;&#x30C8;&#x306B;&#x95A2;&#x4E0E;&#x3059;&#x308B;&#x53EF;&#x80FD;&#x6027;&#x304C;&#x3042;&#x308A;&#x307E;&#x3059;&#x56DE;&#x6587;&#x306E;&#x30B7;&#x30FC;&#x30B1;&#x30F3;&#x30B9;&#x3092;&#x542B;&#x3080; 3 &#x3064;&#x306E;&#x5730;&#x57DF;&#x306E;&#x5305;&#x62EC;&#x7684;&#x306A;&#x69CB;&#x9020;&#x3092;&#x63D0;&#x4F9B;&#x3057;&#x307E;&#x3059;&#x3002;&#x3055;&#x3089;&#x306B;&#x3001;&#x6211;&#x3005; &#x306F;&#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x69D8;&#x7C92;&#x5B50;&#x3068;&#x3092;&#x95A2;&#x9023;&#x4ED8;&#x3051;&#x308B;&#x30EC;&#x30C8;&#x30ED;&#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30DD;&#x30BE;&#x30F3; Ty1 &#x30A2;&#x30F3;&#x30C1;&#x30BB;&#x30F3;&#x30B9;&#x8EE2;&#x5199;&#x306B;&#x57FA;&#x3065;&#x3044;&#x3066;&#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30DD;&#x30BE;&#x30F3; &#x30B3;&#x30F3;&#x30C8;&#x30ED;&#x30FC;&#x30EB;&#x306E;&#x65B0;&#x3057;&#x3044;&#x5F62;&#x614B;&#x306E;&#x5F71;&#x97FF;&#x3092;&#x6C7A;&#x5B9A;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x30A2;&#x30F3;&#x30C1;&#x30BB;&#x30F3;&#x30B9; Rna l1 &#x306E;&#x30EC;&#x30C8;&#x30ED;&#x8EE2;&#x4F4D;&#x3092;&#x963B;&#x5BB3;&#x3059;&#x308B; RNA &#x30B2;&#x30CE;&#x30E0;&#x3092;&#x30A2;&#x30CB;&#x30FC;&#x30EB;&#x3059;&#x308B;&#x306E;&#x3067;&#x306F;&#x306A;&#x304F;&#x30EC;&#x30C8;&#x30ED;&#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30DD;&#x30BE;&#x30F3; Ty1 &#x86CB;&#x767D;&#x8CEA;&#x6A5F;&#x80FD;&#x306E;&#x30BF;&#x30FC;&#x30B2;&#x30C3;&#x30C8;&#x306B;&#x3088;&#x3063;&#x3066;&#x8003;&#x3048;&#x3092;&#x652F;&#x6301;&#x3059;&#x308B;&#x6211;&#x3005; &#x306E;&#x7D50;&#x679C;&#x3002;

&#x30EC;&#x30C8;&#x30ED;&#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30DD;&#x30BE;&#x30F3; Ty1 &#x30EC;&#x30C8;&#x30ED;&#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30DD;&#x30BE;&#x30F3; RNA &#x69CB;&#x9020;&#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x69D8;&#x7C92;&#x5B50;&#x5185;&#x3092;&#x63A2;&#x7D22;&#x3057;&#x307E;&#x3059;&#x3002;

Ty1, Saccharomyces&#xC758; &#xAE34; &#xD130;&#xBBF8;&#xB110; &#xBC18;&#xBCF5; retrotransposon&#xB294; &#xAD6C;&#xC870;&#xC801; &#xBC0F; &#xAE30;&#xB2A5;&#xC801;&#xC73C;&#xB85C; &#xAD00;&#xB828;&#xC774; retroviruses. &#xADF8;&#xB7EC;&#xB098;, &#xC774;&#xB7EC;&#xD55C; retroelements &#xC0AC;&#xC774;&#xC758; &#xCC28;&#xBCC4;&#xD654; &#xCE21;&#xBA74; &#xC624;&#xB798; &#xD130;&#xBBF8;&#xB110; &#xBC18;&#xBCF5; retrotransposons&#xC5D0;&#xC11C; &#xC0AC;&#xC6A9; &#xD558;&#xB294; &#xBCF5;&#xC81C; &#xC804;&#xB7B5;&#xC758; &#xB2E4;&#xC591;&#xC131; &#xC774;&#xB2E4;. &#xBC18;&#xC804; &#xB179;&#xC74C; &#xBC29;&#xC1A1; &#xC81C;&#xC5B4; &#xD558;&#xB294; cis &#xC5F0;&#xAE30; &#xC694;&#xC18C; Ty1 &#xAC8C;&#xB188; RNA&#xC5D0; &#xC788;&#xB294; &#xAC1C; &#xADF8; &#xC9C0;&#xC5ED;&#xC5D0;&#xC11C;&#xC758; &#xAD6C;&#xC870; &#xC870;&#xC9C1; &#xC774;&#xD574; &#xD558;&#xAE30; &#xC6B0;&#xB9AC; chemoenzymatic &#xD504;&#xB85C; &#xBE59; &#xCCB4; &#xC678;&#xC5D0;&#xC11C; &#xC870;&#xB9BD; &#xD558;&#xB294; RNA/tRNA &#xB2E8;&#xC9C0; &#xBC0F; RNA &#xBC14;&#xC774;&#xB7EC;&#xC2A4; &#xAC19;&#xC740; &#xC785;&#xC790;&#xC5D0; &#xC801;&#xC6A9;. &#xB2E4;&#xB978; RNA &#xC0C1;&#xD0DC;&#xB97C; &#xBE44;&#xAD50; &#xD558; &#xC5EC; &#xC6B0;&#xB9AC;&#xC758; &#xBD84;&#xC11D; &#xB1CC;&#xAD00; &#xBC14;&#xC778;&#xB529; &#xC0AC;&#xC774;&#xD2B8;, &#xC18C;&#xC124; pseudoknot &#xB1CC;&#xAD00; &#xBC14;&#xC778;&#xB529; &#xC0AC;&#xC774;&#xD2B8;&#xC5D0; &#xC778;&#xC811; &#xD55C;, RNA dimerization &#xB610;&#xB294; &#xD3EC;&#xC7A5; &#xBC0F; &#xD6C4;&#xBCF4; &#xB2E8;&#xBC31;&#xC9C8; &#xC0C1;&#xD638; &#xC791;&#xC6A9; &#xC0AC;&#xC774;&#xD2B8;&#xC5D0; &#xCC38;&#xC5EC; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4; &#xB41C; &#xC2DC;&#xD000;&#xC2A4;&#xB97C; &#xD3EC;&#xD568; &#xD558;&#xB294; &#xC138; &#xC601;&#xC5ED;&#xC774;&#xC758; &#xD3EC;&#xAD04;&#xC801;&#xC778; &#xAD6C;&#xC870;&#xB97C; &#xC81C;&#xACF5; &#xD569;&#xB2C8;&#xB2E4;. &#xB610;&#xD55C;, &#xC6B0;&#xB9AC; &#xC18C;&#xC124; &#xD615;&#xD0DC;&#xC758; &#xBC14;&#xC774;&#xB7EC;&#xC2A4; &#xAC19;&#xC740; &#xC785;&#xC790;&#xC5D0; &#xC5F0;&#xACB0; Ty1 &#xC13C;&#xC2A4; &#xC131;&#xC801;&#xD45C;&#xC5D0; &#xB530;&#xB77C; transposon &#xCEE8;&#xD2B8;&#xB864;&#xC758; &#xC601;&#xD5A5;&#xC744; &#xD655;&#xC778; &#xD588;&#xC2B5;&#xB2C8;&#xB2E4;. &#xC6B0;&#xB9AC;&#xC758; &#xACB0;&#xACFC; &#xC13C;&#xC2A4; RNAs RNA &#xAC8C;&#xB188;&#xACFC; &#xC5B4; &#xB2D0; &#xB9C1; &#xBCF4;&#xB2E4;&#xB294; Ty1 &#xB2E8;&#xBC31;&#xC9C8; &#xAE30;&#xB2A5;&#xC744; &#xB300;&#xC0C1;&#xC73C;&#xB85C; &#xD558; &#xC5EC; retrotransposition&#xC744; &#xC5B5;&#xC81C; &#xD558;&#xB294; &#xC544;&#xC774;&#xB514;&#xC5B4;&#xB97C; &#xC9C0;&#xC6D0; &#xD569;&#xB2C8;&#xB2E4;.

&#xBC14;&#xC774;&#xB7EC;&#xC2A4; &#xAC19;&#xC740; &#xC785;&#xC790; &#xB0B4;&#xC5D0;&#xC11C; Ty1 retrotransposon RNA &#xAD6C;&#xC870;&#xB97C; &#xD0D0;&#xC0C9;&#xD569;&#xB2C8;&#xB2E4;.

Ty1, un retrotransposon repetici&oacute;n terminal larga de Saccharomyces, estructural y funcionalmente relaciona retrovirus. Sin embargo, un aspecto diferenciador entre estos retroelementos es la diversidad de las estrategias de replicaci&oacute;n utilizadas por retrotransposones repetici&oacute;n terminal larga. Para entender la organizaci&oacute;n estructural de elementos de acci&oacute;n cis presentes en Ty1 RNA gen&oacute;mico de la regi&oacute;n GAG que controlan la transcripci&oacute;n inversa, aplicamos chemoenzymatic sondeo complejos RNA/tRNA montado en vitro y del ARN en part&iacute;culas similares a los virus. Mediante la comparaci&oacute;n de diferentes Estados de RNA, nuestros an&aacute;lisis proporcionan una estructura completa del sitio de cartilla-Uni&oacute;n, una novela pseudoknot adyacente a los sitios de uni&oacute;n de la cartilla, tres regiones con secuencias palindr&oacute;mico que pueden estar involucradas en los sitios de interacci&oacute;n prote&iacute;na dimerizaci&oacute;n o empaquetado y candidato de RNA. Adem&aacute;s, se determin&oacute; el impacto de una forma nueva de transposones control basado en transcripciones antisentidas Ty1 que se asocian con part&iacute;culas similares a los virus. Nuestros resultados apoyan la idea que ARN antisentido inhibe retrotransposition dirigido a la funci&oacute;n de la prote&iacute;na Ty1 en lugar de recocido con el genoma de ARN.

Explorar la estructura de RNA de retrotransposon Ty1 dentro de part&iacute;culas similares a los virus.

Ty1 retrotransposon RNA has the potential to fold into a variety of distinct structures, mutation of which affects retrotransposition frequencies. We show here that one potential functional structure is located at the 5' end of the genome and can assume a pseudoknot conformation. Chemoenzymatic probing of wild-type and mutant mini-Ty1 RNAs supports the existence of such a structure, while molecular genetic analyses show that mutations disrupting pseudoknot formation interfere with retrotransposition, indicating that it provides a critical biological function. These defects are enhanced at higher temperatures. When these mutants are combined with compensatory changes, retrotransposition is restored, consistent with pseudoknot architecture. Analyses of mutants suggest a defect in Ty1 reverse transcription. Collectively, our data allow modeling of a three-dimensional structure for this novel critical cis-acting signal of the Ty1 genome.

Retrotransposon Ty1 RNA contains a 5'-terminal long-range pseudoknot required for efficient reverse transcription.

Ty1 r&eacute;trotransposon que RNA a le potentiel pour plier dans une vari&eacute;t&eacute; de structures distinctes, mutation qui affecte la r&eacute;trotransposition fr&eacute;quences. Nous montrons ici un potentiel fonctionnel structure se trouve &agrave; l&#39;extr&eacute;mit&eacute; 5&#39; du g&eacute;nome et peut prendre une conformation pseudon&oelig;ud. Chimioenzymatique sondage d&#39;ARN sauvage et mutantes mini-Ty1 soutient l&#39;existence d&#39;une telle structure, tandis que des analyses g&eacute;n&eacute;tiques mol&eacute;culaires montrent que des mutations perturbant la formation pseudon&oelig;ud g&ecirc;neront r&eacute;trotransposition, indiquant qu&#39;elle fournit une fonction biologique critique. Ces d&eacute;fauts ont &eacute;t&eacute; am&eacute;lior&eacute;s &agrave; des temp&eacute;ratures plus &eacute;lev&eacute;es. Ces mutants sont conjugu&eacute;es &agrave; des changements compensatoires, r&eacute;trotransposition est compatible avec l&#39;architecture pseudon&oelig;ud, restaur&eacute;e. Analyses de mutants semblent indiquer un d&eacute;faut de transcription inverse Ty1. Collectivement, nos donn&eacute;es permettent la mod&eacute;lisation d&#39;une structure en trois dimensions pour ce nouveau signal d&#39;intramol&eacute;culaires critique du g&eacute;nome Ty1.

R&eacute;trotransposon Ty1 ARN contient un pseudon&oelig;ud &agrave; longue port&eacute;e de 5&#39;-terminal requis pour la transcription inverse efficace.

Retrotrasposoni Ty1 che RNA ha il potenziale per piegare in una variet&agrave; di strutture distinte, mutazione che interessa le frequenze retrotrasposizione. Mostriamo qui che un potenziale funzionale struttura si trova all&#39;estremit&agrave; 5&#39; del genoma e pu&ograve; assumere una conformazione pseudoknot. Chemoenzymatic sondaggio del mutante e del selvaggio-tipo mini-Ty1 RNAs sostiene l&#39;esistenza di tale struttura, mentre le analisi genetiche molecolari mostrano che mutazioni interrompendo la formazione pseudoknot interferiscano con la retrotrasposizione, indicando che esso fornisce una funzione biologica fondamentale. Questi difetti vengono esaltati a temperature pi&ugrave; elevate. Quando questi mutanti sono combinati con modifiche compensative, retrotrasposizione &egrave; ripristinato, coerente con l&#39;architettura pseudoknot. Analisi di mutanti suggeriscono un difetto di trascrizione d&#39;inversione Ty1. Collettivamente, i nostri dati consentono la modellazione di una struttura tridimensionale per questo romanzo critico cis-acting segnale del genoma Ty1.

Retrotrasposoni Ty1 RNA contiene un pseudoknot a lungo raggio 5&#39;-terminale necessaria per efficiente trascrizione d&#39;inversione.

Interaction between the viral protein Rev and the RNA motifs known as Rev response elements (RREs) is required for transport of unspliced and partially spliced human immunodeficiency virus (HIV)-1 and HIV-2 RNAs from the nucleus to the cytoplasm during the later stages of virus replication. A more detailed understanding of these nucleoprotein complexes and the host factors with which they interact should accelerate the development of new antiviral drugs targeting cis-acting RNA regulatory signals. In this communication, the secondary structures of the HIV-2 RRE and two RNA folding precursors have been identified using the SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension) chemical probing methodology together with a novel mathematical approach for determining the secondary structures of RNA conformers present in a mixture. A complementary chemical probing technique was also used to support these secondary structure models, to confirm that the RRE2 RNA undergoes a folding transition and to obtain information about the relative positioning of RRE2 substructures in three dimensions. Our analysis collectively suggests that the HIV-2 RRE undergoes two conformational transitions before assuming the energetically most favorable conformer. The 3D models for the HIV-2 RRE and folding intermediates are also presented, wherein the Rev-binding stem-loops (IIB and I) are located coaxially in the former, which is in agreement with previous models for HIV-1 Rev-RRE binding.

The HIV-2 Rev-response element: determining secondary structure and defining folding intermediates.

Interaction entre la prot&eacute;ine virale Rev et les motifs de RNA appel&eacute;es &eacute;l&eacute;ments de r&eacute;ponse de Rev (RREs) est requise pour le transport d&#39;unspliced et partiellement &eacute;piss&eacute; virus d&#39;immunod&eacute;ficience humaine (VIH) -1 et VIH-2 ARN du noyau vers le cytoplasme pendant les derni&egrave;res phases de la r&eacute;plication du virus. Une compr&eacute;hension plus approfondie de ces complexes nucl&eacute;oprot&eacute;iques et les facteurs de l&#39;h&ocirc;te avec laquelle ils interagissent devrait acc&eacute;l&eacute;rer le d&eacute;veloppement de nouveaux m&eacute;dicaments antiviraux ciblant les signaux r&eacute;gulateurs de RNA intramol&eacute;culaires. Dans la pr&eacute;sente communication, les structures secondaires de la RRE VIH-2 et deux pr&eacute;curseurs de pliage de RNA ont &eacute;t&eacute; identifi&eacute;s &agrave; l&#39;aide de la substance chimique (acylation s&eacute;lective 2&#39;-hydroxyle analys&eacute; par extension d&#39;amorce) de forme m&eacute;thodologie ainsi qu&#39;une nouvelle approche math&eacute;matique permettant de d&eacute;terminer les structures secondaires des conform&egrave;res RNA pr&eacute;sents dans un m&eacute;lange de palpage. Un chimique compl&eacute;mentaire technique de palpage servait aussi pour soutenir ces mod&egrave;les de structure secondaire, pour confirmer que l&#39;ARN RRE2 subit une transition de pliage et d&#39;obtenir des informations sur le positionnement relatif des sous-structures RRE2 en trois dimensions. Collectivement, notre analyse sugg&egrave;re que le VIH-2 RRE subit deux transitions conformationnelles avant d&#39;assumer le conform&egrave;re &eacute;nerg&eacute;tiquement plus favorable. Les mod&egrave;les 3D pour le HIV-2 RRE et pliantes interm&eacute;diaires sont &eacute;galement pr&eacute;sent&eacute;es, dans lequel le Rev-liaison tige-boucle (IIB et moi) est situ&eacute;s coaxialement dans la premi&egrave;re, qui est en accord avec les mod&egrave;les pr&eacute;c&eacute;dents pour la liaison de HIV-1 Rev-RRE.

L&#39;&eacute;l&eacute;ment de HIV-2 Rev-r&eacute;ponse : d&eacute;termination d&#39;une structure secondaire et la d&eacute;finition de pliage interm&eacute;diaires.

Interazione tra la proteina virale Rev ed i motivi di RNA conosciuti come elementi di risposta Rev (RREs) &egrave; richiesto per il trasporto di unspliced e parzialmente impiombato human immunodeficiency virus (HIV) -1 e HIV-2 RNAs dal nucleo al citoplasma durante le successive fasi di replicazione del virus. Una comprensione pi&ugrave; dettagliata di questi complessi nucleoproteine e i fattori di host con cui interagiscono dovrebbe accelerare lo sviluppo di nuovi farmaci antivirali targeting cis-acting segnali di RNA regolatori. In questa comunicazione, sono state identificate le strutture secondarie di RRE l&#39;HIV-2 e due precursori pieghevoli RNA usando il prodotto chimico (selettivo 2&#39;-idrossile acilazione analizzato di estensione dell&#39;iniettore) forma metodologia insieme ad un nuovo approccio matematico per determinare le strutture secondarie di conformeri RNA presenti in una miscela di sondaggio. Un chimico complementare tecnica di sondaggio &egrave; stato utilizzato anche per sostenere questi modelli di struttura secondaria, per confermare che il RNA RRE2 subisce una transizione pieghevole e ottenere informazioni circa il posizionamento relativo di RRE2 sostruzioni in tre dimensioni. Collettivamente, la nostra analisi suggerisce che l&#39;HIV-2 RRE subisce due transizioni conformazionali prima di assumere il conformero energeticamente pi&ugrave; favorevole. I modelli 3D per l&#39;HIV-2 RRE e pieghevole intermedi sono anche presentati, in cui il Rev-associazione stem-loop (IIB e) si trovano coassialmente nel primo caso, che &egrave; in accordo con precedenti modelli per l&#39;associazione di HIV-1 Rev-RRE.

Sabrina Lusvarghi

RT Biochemistry Section,
HIV Drug Resistance Program,
RT Biochemistry Section, HIV Drug Resistance Program

Frederick National Laboratory for Cancer Research

RNA Secondary Structure Prediction Using High-throughput SHAPE

Sabrina Lusvarghi

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}RT Biochemistry Section,HIV Drug Resistance Program,RT Biochemistry Section, HIV Drug Resistance Program

Frederick National Laboratory for Cancer Research

RNA Secondary Structure Prediction Using High-throughput SHAPE

RT Biochemistry Section,
HIV Drug Resistance Program,
RT Biochemistry Section, HIV Drug Resistance Program