real-time-imaging-single-engineered-rna-transcripts-living-cells

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Integrated Dna Technologies, Inc.

Melting temperatures, T(m), were systematically studied for a set of 92 DNA duplex oligomers in a variety of sodium ion concentrations ranging from 69 mM to 1.02 M. The relationship between T(m) and ln [Na(+)] was nonlinear over this range of sodium ion concentrations, and the observed melting temperatures were poorly predicted by existing algorithms. A new empirical relationship was derived from UV melting data that employs a quadratic function, which better models the melting temperatures of DNA duplex oligomers as sodium ion concentration is varied. Statistical analysis shows that this improved salt correction is significantly more accurate than previously suggested algorithms and predicts salt-corrected melting temperatures with an average error of only 1.6 degrees C when tested against an independent validation set of T(m) measurements obtained from the literature. Differential scanning calorimetry studies demonstrate that this T(m) salt correction is insensitive to DNA concentration. The T(m) salt correction function was found to be sequence-dependent and varied with the fraction of G.C base pairs, in agreement with previous studies of genomic and polymeric DNAs. The salt correction function is independent of oligomer length, suggesting that end-fraying and other end effects have little influence on the amount of sodium counterions released during duplex melting. The results are discussed in the context of counterion condensation theory.

Effects of sodium ions on DNA duplex oligomers: improved predictions of melting temperatures.

Cancer patients undergoing triple therapy (CPT-11, 5-fluorouracil, and leucovorin) often present with severe delayed diarrhea as a result of chemotherapy-induced gastrointestinal (GI) toxicity and inflammation. RDP58 is a novel, anti-inflammatory, D-amino acid decapeptide that inhibits the production of tumor necrosis factor alpha, IFN-gamma, and interleukin 12, and has been shown to effectively inhibit clinical symptoms and intestinal inflammation in several rodent models of chemically induced colitis, nonhuman primates with spontaneous colitis, and humans with mild to moderate ulcerative colitis. We evaluated RDP58 as a potential protective agent in chemotherapy-induced GI inflammation. Oral administration of RDP58 significantly decreased the incidence of diarrhea and improved the survival rates of mice treated with toxic doses of CPT-11 or 5-fluorouracil. Histological analysis showed that RDP58 significantly reduced the destruction of the intestinal mucosa by inhibiting local overproduction of tumor necrosis factor alpha, IFN-gamma, and interleukin 12 in vivo. Furthermore, RDP58 administration allowed the maximum tolerated dose of CPT-11 to be doubled in tumor-bearing mice resulting in significantly enhanced primary tumor responses and prolongation of time to relapse without a concomitant increase in GI toxicity. Our results suggest that RDP58 may have clinical utility in cancer therapy by preventing treatment-associated GI toxicity and potentially increasing the effectiveness of chemotherapy.

Oral RDP58 allows CPT-11 dose intensification for enhanced tumor response by decreasing gastrointestinal toxicity.

PIWI-interacting RNAs (piRNAs) are a recently discovered class of small noncoding RNAs that have been detected in human, mouse, rat, zebrafish, and Drosophila genomes. We have utilized a size-directed small-RNA cloning procedure to clone and map more than 300 candidate piRNA-like small RNAs in the genome of the marsupial species Monodelphis domestica. Our results are consistent with those from other species in that the piRNA-like candidate sequences range in size from 28 to 31 nucleotides, show a pronounced preference for uridine at the 5' end, are transcribed from a few large clusters, appear to target transposons, and display virtually no sequence conservation.

PiRNA-like RNAs in the marsupial Monodelphis domestica identify transcription clusters and likely marsupial transposon targets.

&#x5207;&#x5757;&#x673A;&#x57FA;&#x677F;&#x5C0F;&#x5E72;&#x6270; Rna (DsiRNAs) &#x662F;&#x5408;&#x6210;&#x7684; RNA &#x53CC;&#x5DE5;&#xFF0C;&#x7531;&#x5207;&#x5757;&#x673A;&#x6210; 21 mer &#x7269;&#x79CD;&#x8FDB;&#x884C;&#x5904;&#x7406;&#x548C;&#x663E;&#x793A;&#x6539;&#x8FDB;&#x7684;&#x6548;&#x80FD;&#x4F5C;&#x4E3A;&#x89E6;&#x53D1;&#x5668;&#x7684; RNA &#x5E72;&#x6270;&#xFF0C;&#x5C24;&#x5176;&#x5728;&#x4F7F;&#x7528;&#x5728;&#x4F4E;&#x5242;&#x91CF;&#x65F6;&#x3002;&#x5316;&#x5B66;&#x6539;&#x6027;&#x6A21;&#x5F0F;&#xFF0C;&#x662F;&#x4E0E;&#x51E0;&#x4E2A;&#x7EC4;&#x6240;&#x62A5;&#x5C0F;&#x5206;&#x5B50;&#x5E72;&#x6270; Rna &#x7684;&#x9AD8;&#x6D53;&#x5EA6; 21 mer &#x517C;&#x5BB9;&#x3002;&#x4F46;&#x662F;&#xFF0C;&#x4FEE;&#x6539;&#x6A21;&#x5F0F;&#x5C1A;&#x672A;&#x5F97;&#x5230;&#x7814;&#x7A76;&#x7684;&#x5207;&#x5757;&#x673A;&#x57FA;&#x677F;&#x53CC;&#x5DE5;&#x3002;&#x6211;&#x4EEC;&#x56E0;&#x6B64;&#x5408;&#x6210;&#x4E00;&#x7CFB;&#x5217;&#x7684;&#x5316;&#x5B66;&#x4FEE;&#x9970;&#x7684; 27 mer DsiRNAs &#x548C;&#x529F;&#x80FD;&#x6027;&#x7684;&#x6548;&#x80FD;&#x4E0E;&#x76F8;&#x5173;&#x4FEE;&#x6539;&#x6A21;&#x5F0F;&#x3002;&#x4E00;&#x4E9B;&#x4FEE;&#x6539;&#x6A21;&#x5F0F;&#x5927;&#x5927;&#x964D;&#x4F4E;&#x529F;&#x80FD;&#x867D;&#x7136;&#x5176;&#x4ED6;&#x6A21;&#x5F0F;&#x4FDD;&#x6301;&#x9AD8;&#x7684;&#x6548;&#x80FD;&#x3002;&#x89C2;&#x5BDF;&#x5230;&#x5E8F;&#x5217;&#x65B9;&#x9762;&#x7684;&#x5F71;&#x54CD;&#xFF0C;&#x5176;&#x4E2D;&#x76F8;&#x5BF9;&#x6BD2;&#x6027;&#x7684;&#x6539;&#x9020;&#x6A21;&#x5F0F;&#x7AD9;&#x70B9;&#x4E4B;&#x95F4;&#x5404;&#x4E0D;&#x76F8;&#x540C;&#x3002;&#x4FEE;&#x6539;&#x6A21;&#x5F0F;&#x6D89;&#x53CA;&#x4EA4;&#x66FF; 2&#39;-O-&#x7532;&#x57FA; RNA &#x57FA;&#x5730;&#x53D1;&#x8FBE;&#x56FD;&#x5BB6;&#xFF0C;&#x4E00;&#x822C;&#x4FDD;&#x7559;&#x9AD8;&#x70C8;&#x5EA6;&#x5728;&#x4E0D;&#x540C;&#x7684;&#x5730;&#x70B9;&#xFF0C;&#x4E0D;&#x540C;&#x7684;&#x57FA;&#x56E0;&#xFF0C;&#x5728;&#x6D4B;&#x8BD5;&#x65F6;&#x9003;&#x907F;&#x7684;&#x5148;&#x5929;&#x514D;&#x75AB;&#x7CFB;&#x7EDF;&#xFF0C;&#x6FC0;&#x6D3B;&#x5E76;&#x63D0;&#x9AD8;&#x8840;&#x6E05;&#x4E2D;&#x7684;&#x7A33;&#x5B9A;&#x6027;&#x3002;

&#x5316;&#x5B66;&#x4FEE;&#x9970;&#x6A21;&#x5F0F;&#x4E0E;&#x9AD8;&#x6D53;&#x5EA6;&#x5207;&#x5757;&#x673A;&#x886C;&#x5E95;&#x5C0F;&#x5E72;&#x6270; Rna &#x517C;&#x5BB9;&#x3002;

Dicer-substrate small interfering RNAs (DsiRNAs) are synthetic RNA duplexes that are processed by Dicer into 21-mer species and show improved potency as triggers of RNA interference, particularly when used at low dose. Chemical modification patterns that are compatible with high potency 21-mer small interfering RNAs have been reported by several groups. However, modification patterns have not been studied for Dicer-substrate duplexes. We therefore synthesized a series of chemically modified 27-mer DsiRNAs and correlated modification patterns with functional potency. Some modification patterns profoundly reduced function although other patterns maintained high potency. Effects of sequence context were observed, where the relative potency of modification patterns varied between sites. A modification pattern involving alternating 2'-O-methyl RNA bases was developed that generally retains high potency when tested in different sites in different genes, evades activation of the innate immune system, and improves stability in serum.

Chemical modification patterns compatible with high potency dicer-substrate small interfering RNAs.

Dicer-substrat de petits ARN interf&#xE9;rents (DsiRNAs) sont synth&#xE9;tiques duplex ARN qui sont trait&#xE9;es par Dicer en 21-mer esp&#xE8;ces et de montrer la puissance am&#xE9;lior&#xE9;e comme des d&#xE9;clencheurs de l&#x26;#39;interf&#xE9;rence ARN, en particulier lorsqu&#x26;#39;il est utilis&#xE9; &#xE0; faible dose. Sch&#xE9;mas de modification chimique qui sont compatibles avec la puissance &#xE9;lev&#xE9;e de 21 mer de petits ARN interf&#xE9;rents ont &#xE9;t&#xE9; rapport&#xE9;s par plusieurs groupes. Toutefois, les tendances de modification n&#x26;#39;ont pas &#xE9;t&#xE9; &#xE9;tudi&#xE9;s pour Dicer-substrat duplex. Nous avons donc synth&#xE9;tis&#xE9; une s&#xE9;rie de chimiquement modifi&#xE9;es 27-mer et des mod&#xE8;les de modification DsiRNAs corr&#xE9;l&#xE9;s avec une puissance fonctionnelle. Certains mod&#xE8;les de modification profonde diminution de la fonction m&#xEA;me si les mod&#xE8;les d&#x26;#39;autres maintenue forte puissance. Effets de contexte de s&#xE9;quence ont &#xE9;t&#xE9; observ&#xE9;es, o&#xF9; la puissance relative des modes de modification vari&#xE9; entre les sites. Un motif de modification impliquant alternance 2&#x26;#39;-O-m&#xE9;thyle bases de l&#x26;#39;ARN a &#xE9;t&#xE9; d&#xE9;velopp&#xE9; qui conserve g&#xE9;n&#xE9;ralement forte puissance lorsqu&#x26;#39;il est test&#xE9; dans des sites diff&#xE9;rents dans diff&#xE9;rents g&#xE8;nes, &#xE9;lude l&#x26;#39;activation du syst&#xE8;me immunitaire inn&#xE9;, et am&#xE9;liore la stabilit&#xE9; dans le s&#xE9;rum.

Mod&#xE8;les de modification chimique compatible avec forte puissance Dicer-substrat de petits ARN interf&#xE9;rents

Dicer-Substrat kleine interferierender RNAs (DsiRNAs) sind synthetische RNA-Duplex, die verbesserte Potenz als Ausl&ouml;ser der RNA-Interferenz, zeigen, insbesondere wenn bei niedrigen Dosis verwendet und von Dicer in 21-Mer Arten verarbeitet werden. Chemische Modifikation Muster sind, die kompatibel mit hoher Potenz 21-Mer, die kleine interferierender RNAs von mehreren Gruppen gemeldet wurden. &Auml;nderung Muster wurden jedoch nicht f&uuml;r Dicer-Substrat Duplices untersucht. Wir haben daher synthetisiert eine Reihe von chemisch modifizierten 27-Mer DsiRNAs und &Auml;nderung Muster mit funktionalen Potenz korreliert. Einige &Auml;nderung Muster reduziert zutiefst Funktion, obwohl andere Muster hoher Potenz erhalten. Auswirkungen der Sequenz Kontext wurden beobachtet, wo die relative Wirksamkeit der &Auml;nderung Muster zwischen Standorten variiert. Eine &Auml;nderung Muster mit abwechselnd 2&#39;-O-Methyl-RNA-Basen entwickelt wurde, die im allgemeinen hohe Potenz, wenn an unterschiedlichen Standorten in verschiedenen Genen getestet beh&auml;lt entzieht Aktivierung des angeborenen Immunsystems und verbessert die Stabilit&auml;t im Serum.

Chemische Modifikation Muster mit hoher Potenz Dicer-Substrat st&ouml;ren kleine RNAs kompatibel.

Cubettatrice-substrato piccolo RNAs d&#39;interferenza (DsiRNAs) sono duplex RNA sintetico che vengono elaborati da Dicer in mer 21 specie e visualizza potenza migliorata come trigger di interferenza del RNA, specialmente quando utilizzato a basse dosi. Modelli di modificazione chimica che sono compatibili con alta potenza 21-mer che small interfering RNAs sono stati segnalati da diversi gruppi. Modelli di modifica, tuttavia, non sono stati studiati per Duplex Dicer-substrato. Pertanto abbiamo sintetizzato una serie di 27-mer chimicamente modificati DsiRNAs e correlato modelli di modifica con potenza funzionale. Alcuni modelli di modifica profondamente funzione ridotta anche se altri modelli mantenuto ad alta potenza. Sono stati osservati effetti di contesto di sequenza, dove la potenza dei modelli di modifica relativa varia tra i siti. Un modello di modifica che coinvolgono alternando 2&#39;--O metil RNA basi &egrave; stato sviluppato che generalmente mantiene alta potenza quando testati in diversi siti in diversi geni, evade l&#39;attivazione del sistema immunitario innato e migliora la stabilit&agrave; nel siero.

Modelli di modificazione chimica compatibili con alta potenza dicer-substrato small interfering RNAs.

&#x30C0;&#x30A4;&#x30B5;&#x30FC;&#x57FA;&#x677F;&#x4F4E;&#x5206;&#x5B50;&#x5E72;&#x6E09;RNA&#xFF08;DsiRNAs&#xFF09;&#x306F;21-mer&#x306E;&#x7A2E;&#x306B;&#x30C0;&#x30A4;&#x30B5;&#x30FC;&#x306B;&#x3088;&#x3063;&#x3066;&#x51E6;&#x7406;&#x3055;&#x308C;&#x3001;&#x4F4E;&#x7528;&#x91CF;&#x3067;&#x4F7F;&#x7528;&#x3059;&#x308B;&#x5834;&#x5408;&#x306F;&#x7279;&#x306B;&#x3001;RNA&#x5E72;&#x6E09;&#x306E;&#x30C8;&#x30EA;&#x30AC;&#x30FC;&#x3068;&#x3057;&#x3066;&#x6539;&#x826F;&#x3055;&#x308C;&#x305F;&#x52B9;&#x529B;&#x3092;&#x793A;&#x3057;&#x3066;&#x3044;&#x305F;&#x5408;&#x6210;RNA&#x4E8C;&#x91CD;&#x9396;&#x3067;&#x3042;&#x308B;&#x3002;&#x9AD8;&#x52B9;&#x529B;&#x306E;21-mer&#x306E;&#x4F4E;&#x5206;&#x5B50;&#x5E72;&#x6E09;RNA&#x3068;&#x4E92;&#x63DB;&#x6027;&#x306E;&#x3042;&#x308B;&#x5316;&#x5B66;&#x4FEE;&#x98FE;&#x30D1;&#x30BF;&#x30FC;&#x30F3;&#x306F;&#x3044;&#x304F;&#x3064;&#x304B;&#x306E;&#x30B0;&#x30EB;&#x30FC;&#x30D7;&#x306B;&#x3088;&#x308A;&#x5831;&#x544A;&#x3055;&#x308C;&#x3066;&#x3044;&#x308B;&#x3002;&#x3057;&#x304B;&#x3057;&#x3001;&#x4FEE;&#x6B63;&#x30D1;&#x30BF;&#x30FC;&#x30F3;&#x306F;&#x30C0;&#x30A4;&#x30B5;&#x30FC;&#x57FA;&#x677F;&#x4E8C;&#x91CD;&#x9396;&#x306E;&#x305F;&#x3081;&#x306B;&#x691C;&#x8A0E;&#x3055;&#x308C;&#x3066;&#x3044;&#x306A;&#x3044;&#x3002;&#x305D;&#x3053;&#x3067;&#x6211;&#x3005;&#x306F;&#x6A5F;&#x80FD;&#x7684;&#x306A;&#x52B9;&#x529B;&#x3092;&#x6709;&#x3059;&#x308B;&#x5316;&#x5B66;&#x7684;&#x306B;&#x4FEE;&#x98FE;&#x3055;&#x308C;&#x305F;27-mer&#x306E;DsiRNAs&#x3068;&#x76F8;&#x95A2;&#x306E;&#x5909;&#x66F4;&#x4E00;&#x9023;&#x306E;&#x30D1;&#x30BF;&#x30FC;&#x30F3;&#x3092;&#x5408;&#x6210;&#x3057;&#x305F;&#x3002;&#x4ED6;&#x306E;&#x30D1;&#x30BF;&#x30FC;&#x30F3;&#x306F;&#x3001;&#x9AD8;&#x3044;&#x52B9;&#x529B;&#x3092;&#x7DAD;&#x6301;&#x3057;&#x305F;&#x3082;&#x306E;&#x306E;&#x3001;&#x3044;&#x304F;&#x3064;&#x304B;&#x306E;&#x5909;&#x66F4;&#x30D1;&#x30BF;&#x30FC;&#x30F3;&#x306F;&#x6DF1;&#x304F;&#x95A2;&#x6570;&#x3092;&#x6E1B;&#x5C11;&#x3055;&#x305B;&#x305F;&#x3002;&#x5909;&#x66F4;&#x30D1;&#x30BF;&#x30FC;&#x30F3;&#x306E;&#x76F8;&#x5BFE;&#x7684;&#x52B9;&#x529B;&#x200B;&#x200B;&#x306F;&#x3001;&#x30B5;&#x30A4;&#x30C8;&#x9593;&#x3067;&#x5909;&#x5316;&#x304C;&#x30B7;&#x30FC;&#x30B1;&#x30F3;&#x30B9;&#x30B3;&#x30F3;&#x30C6;&#x30AD;&#x30B9;&#x30C8;&#x306E;&#x52B9;&#x679C;&#x306F;&#x3001;&#x89B3;&#x5BDF;&#x3055;&#x308C;&#x305F;&#x3002; 2&#x26;#39;-O-&#x30E1;&#x30C1;&#x30EB;RNA&#x5869;&#x57FA;&#x3092;&#x4EA4;&#x4E92;&#x306B;&#x4F34;&#x3046;&#x5909;&#x66F4;&#x306E;&#x30D1;&#x30BF;&#x30FC;&#x30F3;&#x306F;&#x7570;&#x306A;&#x308B;&#x907A;&#x4F1D;&#x5B50;&#x306E;&#x7570;&#x306A;&#x308B;&#x30B5;&#x30A4;&#x30C8;&#x3067;&#x8A66;&#x9A13;&#x3057;&#x305F;&#x3068;&#x304D;&#x3001;&#x4E00;&#x822C;&#x7684;&#x306B;&#x9AD8;&#x3044;&#x52B9;&#x529B;&#x3092;&#x4FDD;&#x6301;&#x3057;&#x3066;&#x3044;&#x308B;&#x958B;&#x767A;&#x3055;&#x308C;&#x305F;&#x3001;&#x81EA;&#x7136;&#x514D;&#x75AB;&#x7CFB;&#x306E;&#x6D3B;&#x6027;&#x5316;&#x3092;&#x5FCC;&#x907F;&#x3057;&#x3001;&#x8840;&#x6E05;&#x4E2D;&#x306E;&#x5B89;&#x5B9A;&#x6027;&#x3092;&#x5411;&#x4E0A;&#x3055;&#x305B;&#x307E;&#x3059;&#x3002;

&#x30CF;&#x30A4;&#x30DD;&#x30C0;&#x30A4;&#x30B5;&#x30FC;&#x57FA;&#x677F;&#x4F4E;&#x5206;&#x5B50;&#x5E72;&#x6E09;RNA&#x306E;&#x4E92;&#x63DB;&#x5316;&#x5B66;&#x4FEE;&#x98FE;&#x30D1;&#x30BF;&#x30FC;&#x30F3;

Dicer &#xAE30;&#xD310; &#xC791;&#xC740; &#xBC29;&#xD574; RNAs (DsiRNAs)&#xB294; &#xD569;&#xC131; RNA duplexes 21 &#xBA54; &#xC885;&#xC5D0; Dicer&#xC5D0; &#xC758;&#xD574; &#xCC98;&#xB9AC; &#xB418; &#xACE0; &#xB0AE;&#xC740; &#xBCF5;&#xC6A9;&#xB7C9;&#xC5D0; &#xC0AC;&#xC6A9; &#xD558;&#xB294; &#xACBD;&#xC6B0;&#xC5D0; &#xD2B9;&#xD788; RNA &#xAC04;&#xC12D;&#xC758; &#xD2B8;&#xB9AC;&#xAC70;&#xB85C; &#xD5A5;&#xC0C1; &#xB41C; &#xD798;&#xC744; &#xBCF4;&#xC5EC;. &#xD654;&#xD559; &#xC218;&#xC815; &#xD328;&#xD134;&#xC744; &#xB192;&#xC740; &#xD798; 21 &#xBA54; &#xB974; &#xC791;&#xC740; &#xBC29;&#xD574; RNAs &#xC5EC;&#xB7EC; &#xADF8;&#xB8F9;&#xC5D0; &#xC758;&#xD574; &#xBCF4;&#xACE0; &#xB418;&#xC5C8;&#xC2B5;&#xB2C8;&#xB2E4;&#xC640; &#xD638;&#xD658; &#xB429;&#xB2C8;&#xB2E4;. &#xADF8;&#xB7EC;&#xB098;, &#xC218;&#xC815; &#xD328;&#xD134; Dicer &#xAE30;&#xD310; duplexes &#xC704;&#xD574; &#xACF5;&#xBD80; &#xD558;&#xC9C0; &#xC54A;&#xC740;. &#xB530;&#xB77C;&#xC11C; &#xD654;&#xD559;&#xC801; &#xC218;&#xC815;&#xB41C; 27 &#xBA54; &#xB974; DsiRNAs &#xC2DC;&#xB9AC;&#xC988;&#xB97C; &#xD569;&#xC131; &#xD558; &#xACE0; &#xAE30;&#xB2A5;&#xC801; &#xD6A8;&#xB2A5; &#xC218;&#xC815; &#xD328;&#xD134; &#xC5F0;&#xAD00;. &#xB2E4;&#xB978; &#xD328;&#xD134; &#xC720;&#xC9C0; &#xB192;&#xC740; &#xD798; &#xC788;&#xC9C0;&#xB9CC; &#xC77C;&#xBD80; &#xC218;&#xC815; &#xD328;&#xD134; &#xBFCC;&#xB9AC;&#xAE4A;&#xC740; &#xD568;&#xC218;&#xB97C; &#xAC10;&#xC18C;. &#xC218;&#xC815; &#xD328;&#xD134;&#xC758; &#xC0C1;&#xB300;&#xC801; &#xD6A8;&#xB2A5; &#xC0AC;&#xC774;&#xD2B8; &#xAC04;&#xC5D0; &#xB2E4;&#xC591; &#xD55C; &#xD6A8;&#xACFC;&#xC758; &#xC2DC;&#xD000;&#xC2A4; &#xCEE8;&#xD14D;&#xC2A4;&#xD2B8; &#xAD00;&#xCC30; &#xD588;&#xB2E4;. &#xC77C;&#xBC18;&#xC801;&#xC73C;&#xB85C; &#xC11C;&#xB85C; &#xB2E4;&#xB978; &#xC720;&#xC804;&#xC790;&#xC5D0; &#xB2E4;&#xB978; &#xC0AC;&#xC774;&#xD2B8;&#xC5D0;&#xC11C; &#xD14C;&#xC2A4;&#xD2B8; &#xD588;&#xC744; &#xB54C; &#xB192;&#xC740; &#xD798;&#xC744; &#xC720;&#xC9C0; &#xD558;&#xB294; &#xC218;&#xC815; &#xD328;&#xD134; &#xAC1C;&#xBC1C; &#xB418;&#xC5C8;&#xB2E4; &#xBA54; 2&#39;-O-&#xD2F8; RNA &#xAE30;&#xC9C0;&#xB97C; &#xBC88;&#xAC08;&#xC544;&#xAC00; &#xBA70; &#xAD00;&#xB828; &#xB41C; &#xD0C0;&#xACE0; &#xB09C; &#xBA74;&#xC5ED; &#xC2DC;&#xC2A4;&#xD15C;&#xC758; &#xD65C;&#xC131;&#xD654;&#xB97C; evades &#xBC0F; &#xD608; &#xCCAD;&#xC5D0;&#xC11C; &#xC548;&#xC815;&#xC131;&#xC744; &#xD5A5;&#xC0C1; &#xC2DC;&#xD0B5;&#xB2C8;&#xB2E4;.

&#xD654;&#xD559; &#xC218;&#xC815; &#xD328;&#xD134; &#xB192;&#xC740; &#xD798; dicer &#xAE30;&#xD310; &#xC791;&#xC740; &#xBC29;&#xD574; Rnas&#xC640; &#xD638;&#xD658;.

Sustrato Dicer-peque&#xF1;os RNAs de interferencia (DsiRNAs) son sint&#xE9;ticos ARN d&#xFA;plex que son procesados &#x200B;&#x200B;por Dicer en 21-mer especies y muestran la potencia mejorada como factores desencadenantes de la interferencia de ARN, en particular cuando se usa en dosis bajas. Los patrones de modificaci&#xF3;n qu&#xED;mica que son compatibles con alta potencia de 21 mer peque&#xF1;os RNAs de interferencia han sido reportados por varios grupos. Sin embargo, los patrones de las modificaciones que no han sido estudiados por d&#xFA;plex sustrato Dicer. Por lo tanto, sintetizado una serie de modificaciones qu&#xED;micas 27-mer DsiRNAs y patrones correlacionados con la potencia de modificaci&#xF3;n funcional. Algunos patrones de modificaci&#xF3;n profunda disminuci&#xF3;n de la funci&#xF3;n, aunque otros patrones mantiene alta potencia. Efectos de la secuencia contexto se observaron, donde la potencia relativa de los patrones de modificaci&#xF3;n vari&#xF3; entre los sitios. Un patr&#xF3;n de modificaci&#xF3;n que implica alterna 2&#x26;#39;-O-metil bases de ARN fue desarrollado que retiene generalmente de alta potencia cuando se prob&#xF3; en sitios diferentes en diferentes genes, elude la activaci&#xF3;n del sistema inmune innato, y mejora la estabilidad en el suero.

Qu&#xED;micos patrones de modificaci&#xF3;n Compatible con alta potencia de sustrato Dicer-peque&#xF1;os RNAs de interferencia

The involvement of small RNAs in cotton fiber development is under explored. The objective of this work was to directly clone, annotate, and analyze small RNAs of developing ovules to reveal the candidate small interfering RNA/microRNAs involved in cotton ovule and fiber development.

Small RNA regulation of ovule development in the cotton plant, G. hirsutum L.

The "RNA revolution" that started at the end of the 20th century with the discovery of post-transcriptional gene silencing and its mechanism via RNA interference (RNAi) placed tiny 21-24 nucleotide long noncoding RNAs (ncRNAs) in the forefront of biology as one of the most important regulatory elements in a host of physiologic processes. The discovery of new classes of ncRNAs including endogenous small interfering RNAs, microRNAs, and PIWI-interacting RNAs is a hallmark in the understanding of RNA-dependent gene regulation. New generation high-throughput sequencing technologies further accelerated the studies of this "tiny world" and provided their global characterization and validation in many biological systems with sequenced genomes. Nevertheless, for the many "yet-unsequenced" plant genomes, the discovery of small RNA world requires in vitro cloning from purified cellular RNAs. Thus, reproducible methods for in vitro small RNA cloning are of paramount importance and will remain so into the foreseeable future. In this paper, we present a description of existing small RNA cloning methods as well as next-generation sequencing methods that have accelerated this research along with a description of the application of one in vitro cloning method in an initial small RNA survey in the "still unsequenced" allotetraploid cotton genome.

Methodologies for in vitro cloning of small RNAs and application for plant genome(s).

MicroRNAs (miRNAs) are an important class of posttranscriptional gene expression regulators. In the course of mapping novel marsupial-specific miRNAs in the genome of the gray short-tailed opossum, Monodelphis domestica, we encountered a cluster of 39 actual and potential miRNAs spanning 102 kb of the X chromosome. Analysis of the cluster revealed that 37 of the 39 miRNAs are predicted to form thermodynamically stable hairpins, and at least 3 members have been directly cloned from M. domestica tissues. The sequence characteristics of these miRNAs suggest that they all descended from a single common ancestor. Further, 2 distinct families appear to have diversified from the ancestral sequence through different duplication mechanisms: one through a series of simple tandem duplications and the other through a recurrent transposon-mediated duplication process.

An X chromosome microRNA cluster in the marsupial species Monodelphis domestica.

Systematic evolution of ligands by exponential enrichment (SELEX) is a powerful in vitro selection process used for over 2 decades to identify oligonucleotide sequences (aptamers) with desired properties (usually high affinity for a protein target) from randomized nucleic acid libraries. In the case of RNA aptamers, several highly complex RNA libraries have been described with RNA sequences ranging from 71 to 81 nucleotides (nt) in length. In this study, we used high-throughput sequencing combined with bioinformatics analysis to thoroughly examine the nucleotide composition of the sequence pools derived from several selections that employed an RNA library (Sel2N20) with an abbreviated variable region. The Sel2N20 yields RNAs 51 nt in length, which unlike longer RNAs, are more amenable to large-scale chemical synthesis for therapeutic development. Our analysis revealed a consistent and early bias against inclusion of adenine, resulting in aptamers with lower predicted minimum free energies (ΔG) (higher structural stability). This bias was also observed in control, "nontargeted" selections in which the partition step (against the target) was omitted, suggesting that the bias occurred in 1 or more of the amplification and propagation steps of the SELEX process.

Nucleotide bias observed with a short SELEX RNA aptamer library.

Artificial RNA reagents such as small interfering RNAs (siRNAs) and aptamers often must be chemically modified for optimal effectiveness in environments that include ribonucleases. Mycoplasmas are common bacterial contaminants of mammalian cell cultures that are known to produce ribonucleases. Here we describe the rapid degradation of nuclease-stabilized RNA oligonucleotides in a human embryonic kidney 293 (HEK) cell culture contaminated with Mycoplasma fermentans, a common species of mycoplasma. RNA with 2'-fluoro- or 2'-O-methyl- modified pyrimidines was readily degraded in conditioned media from this culture, but was stable in conditioned media from uncontaminated HEK cells. RNA completely modified with 2'-O-methyls was not degraded in the mycoplasma-contaminated media. RNA zymogram analysis of conditioned culture media and material centrifuged from the media revealed several distinct protein bands (ranging from 30 to 68 kDa) capable of degrading RNA with 2'-fluoro- or 2'-O-methyl-modified pyrimidines. Finally, the mycoplasma-associated nuclease was detected in material centrifuged from the contaminated culture supernatants in as little as 15 minutes with an RNA oligo-containing 2'-O-methyl-modified pyrimidines and labeled with a 5'-fluorescein amidite (FAM) and 3'-quencher. These results suggest that mycoplasma contamination may be a critical confounding variable for cell culture experiments involving RNA-based reagents, with particular relevance for applications involving naked RNA (e.g., aptamer-siRNA chimeras).

Degradation of nuclease-stabilized RNA oligonucleotides in Mycoplasma-contaminated cell culture media.

The aims of this study were to investigate the functions of muscle torque and its relation to other torque components during sprinting stance and swing phases. Three-dimensional kinematics and ground reaction force data were collected from eight elite male sprinters performing maximal-effort sprinting on a synthetic track. Intersegmental dynamics approach (ISD) was used to quantify lower extremity joint torque and their components during one gait cycle of the maximal speed phase during sprinting. Specifically, a modified version of the ISD was used to determine the relationship among the active muscle torque (MST), passive motion-dependent torque (MDT), ground reaction torque (EXT), gravitational torque (GTT), and net joint torque (NET) during stance and swing phases. The contribution of each torque component to lower extremity joint motion was quantified. Our results revealed that the active MST functioned to counteract EXT during stance phase. EXT acted to accelerate knee extension and hip flexion, meanwhile the muscles across these joints produced flexion torque at the knee and extension torque at the hip. During swing phase, MDT at the knee and hip joints was mainly produced by leg angular acceleration which was very significant at the moment when leg swing from forward to backward, active MST counterbalanced the effect of MDT. In summary, muscle torque functions mainly to push the ground to counter ground reaction force for controlling the movement during stance phase. However, the role of muscle torque changes during swing phase to mainly counteract the effect of MDT to control the movement direction of the lower extremity at both the hip and knee joints.

Segment-interaction and its relevance to the control of movement during sprinting.

Live mRNA detection allows real time monitoring of specific transcripts and genetic alterations. The main challenge of live genetic detection is overcoming the high background generated by unbound probes and reaching high level of specificity with minimal off target effects. The use of Fluorescence Resonance Energy Transfer (FRET) probes allows differentiation between bound and unbound probes thus decreasing background. Probe specificity can be optimized by adjusting the length and through use of chemical modifications that alter binding affinity. Herein, we report the use of two oligonucleotide FRET probe system to detect a single nucleotide polymorphism (SNP) in murine Hras mRNA, which is associated with malignant transformations. The FRET oligonucleotides were modified with phosphorothioate (PS) bonds, 2'OMe RNA and LNA residues to enhance nuclease stability and improve SNP discrimination. Our results show that a point mutation in Hras can be detected in endogenous RNA of living cells. As determined by an Acceptor Photobleaching method, FRET levels were higher in cells transfected with perfect match FRET probes whereas a single mismatch showed decreased FRET signal. This approach promotes in vivo molecular imaging methods and could further be applied in cancer diagnosis and theranostic strategies.

SNP detection in mRNA in living cells using allele specific FRET probes.

[This corrects the article on p. e72389 in vol. 8.].

Correction: SNP Detection in mRNA in Living Cells Using Allele Specific FRET Probes.

The relationship between RNA expression and cell function can often be difficult to decipher due to the presence of both temporal and sub-cellular processing of RNA. These intricacies of RNA regulation can often be overlooked when only acquiring global measurements of RNA expression. This has led to development of several tools that allow for the real-time imaging of individual engineered RNA transcripts in living cells. Here, we describe a new technique that utilizes an oligonucleotide-based probe, ratiometric bimolecular beacon (RBMB), to image RNA transcripts that were engineered to contain 96-tandem repeats of the RBMB target sequence in the 3'-untranslated region. Binding of RBMBs to the target RNA resulted in discrete bright fluorescent spots, representing individual transcripts, that could be imaged in real-time. Since RBMBs are a synthetic probe, the use of photostable, bright, and red-shifted fluorophores led to a high signal-to-background. RNA motion was readily characterized by both mean squared displacement and moment scaling spectrum analyses. These analyses revealed clear examples of directed, Brownian, and subdiffusive movements.

Imaging the directed transport of single engineered RNA transcripts in real-time using ratiometric bimolecular beacons.

Technologies that enable the rapid detection and localization of bacterial infections in living animals could address an unmet need for infectious disease diagnostics. We describe a molecular imaging approach for the specific, noninvasive detection of S. aureus based on the activity of the S. aureus secreted nuclease, micrococcal nuclease (MN). Several short synthetic oligonucleotides, rendered resistant to mammalian serum nucleases by various chemical modifications and flanked with a fluorophore and quencher, were activated upon degradation by purified MN and in S. aureus culture supernatants. A probe consisting of a pair of deoxythymidines flanked by several 2'-O-methyl-modified nucleotides was activated in culture supernatants of S. aureus but not in culture supernatants of several other pathogenic bacteria. Systemic administration of this probe to mice bearing S. aureus muscle infections resulted in probe activation at the infection sites in an MN-dependent manner. This new bacterial imaging approach has potential clinical applicability for infections with S. aureus and several other medically important pathogens.

Lingyan Huang

Integrated Dna Technologies, Inc.

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons