Name: true gene silencing: screening of a heptamer-type small guide rna library for potential cancer therapeutic agents
Uploaded: 2016-06-02T14:00:00
Description: Watch this Scientific Journal Video about TRUE Gene Silencing: Screening of a Heptamer-type Small Guide RNA Library for Potential Cancer Therapeutic Agents at JoVE.com

这项工作是由适应性和无缝技术转移计划，通过目标驱动的研发，日本科学技术振兴机构，从促进和互助公司为日本的民办学校学研究促进基金JSPS KAKENHI授权号码24300342和15H04313的支持， 。

1.一个七聚物型的构建因组库<ol><li>除非满量程库是要构造选择16,384（4 7）7-核苷酸序列的子集。 注：序列可以是无规和/或设计为靶向特定的细胞的RNA。对于后者，发现发夹结构类似的tRNA的T形臂与立即的发夹结构4,10的下游7-核苷酸序列互补的合适的计算机程序和/或视觉，和选择的序列的辅助靶RNA， 17,18。 </li><li>合成每个选定七聚体的作为完全2&#39;-O-甲基化?...

<ol>
	<li>Scherer, L., Rossi, J. J., Morris, K. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+potential+of+RNA-mediated+control+of+gene+expression:+Options+and+designs.">Therapeutic potential of RNA-mediated control of gene expression: Options and designs.</a> RNA and the Regulation of Gene Expression: A hidden layer of complexity. , 201-226 (2008).</li><li>Nashimoto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conversion+of+mammalian+tRNA+3&#8242;+processing+endoribonuclease+to+four-base-recognizing+RNA+cutters.">Conversion of mammalian tRNA 3&#8242; processing endoribonuclease to four-base-recognizing RNA cutters.</a> Nucleic Acids Res. 23, 3642-3647 (1995).</li><li>Nashimoto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+cleavage+of+target+RNAs+from+HIV-1+with+5&#8242;+half+tRNA+by+mammalian+tRNA+3&#8242;+processing+endoribonuclease.">Specific cleavage of target RNAs from HIV-1 with 5&#8242; half tRNA by mammalian tRNA 3&#8242; processing endoribonuclease.</a> RNA. 2, 523-534 (1996).</li><li>Nashimoto, M., Geary, S., Tamura, M., Kasper, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+heptamers+that+directs+RNA+cleavage+by+mammalian+tRNA+3&#8242;+processing+endoribonuclease.">RNA heptamers that directs RNA cleavage by mammalian tRNA 3&#8242; processing endoribonuclease.</a> Nucleic Acids Res. 26, 2565-2571 (1998).</li><li>Nashimoto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anomalous+RNA+substrates+for+mammalian+tRNA+3&#8242;+processing+endoribonuclease.">Anomalous RNA substrates for mammalian tRNA 3&#8242; processing endoribonuclease.</a> FEBS Lett. 472, 179-186 (2000).</li><li>Takaku, H., Minagawa, A., Takagi, M., Nashimoto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+candidate+prostate+cancer+susceptibility+gene+encodes+tRNA+3&#8242;+processing+endoribonuclease.">A candidate prostate cancer susceptibility gene encodes tRNA 3&#8242; processing endoribonuclease.</a> Nucleic Acids Res. 31, 2272-2278 (2003).</li><li>Takaku, H., Minagawa, A., Takagi, M., Nashimoto, 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+N-terminal+half-domain+of+the+long+form+of+tRNase+Z+is+required+for+the+RNase+65+activity.">The N-terminal half-domain of the long form of tRNase Z is required for the RNase 65 activity.</a> Nucleic Acids Res. 32, 4429-4438 (2004).</li><li>Takaku, H., Minagawa, A., Takagi, M., Nashimoto, M. <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+four-base-recognizing+RNA+cutter+that+can+remove+the+single+3&#8242;+terminal+nucleotides+from+RNA+molecules.">A novel four-base-recognizing RNA cutter that can remove the single 3&#8242; terminal nucleotides from RNA molecules.</a> Nucleic Acids Res. 32, e91 (2004).</li><li>Shibata, H. S., Takaku, H., Takagi, M., Nashimoto, 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+T+loop+structure+is+dispensable+for+substrate+recognition+by+tRNase+ZL.">The T loop structure is dispensable for substrate recognition by tRNase ZL.</a> J. Biol. Chem. 280, 22326-22334 (2005).</li><li>Tamura, M., Nashimoto, C., Miyake, N., Daikuhara, Y., Ochi, K., Nashimoto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+mRNA+cleavage+by+3&#8242;+tRNase+under+the+direction+of+2&#8242;-O-methyl+RNA+heptamers.">Intracellular mRNA cleavage by 3&#8242; tRNase under the direction of 2&#8242;-O-methyl RNA heptamers.</a> Nucleic Acids Res. 31, 4354-4360 (2003).</li><li>Habu, Y., 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=Inhibition+of+HIV-1+gene+expression+by+retroviral+vector-mediated+small-guide+RNAs+that+direct+specific+RNA+cleavage+by+tRNase+ZL.">Inhibition of HIV-1 gene expression by retroviral vector-mediated small-guide RNAs that direct specific RNA cleavage by tRNase ZL.</a> Nucleic Acids Res. 33, 235-243 (2005).</li><li>Nakashima, A., 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=Gene-silencing+by+the+tRNA+maturase+tRNase+ZL+under+the+direction+of+small+guide+RNA.">Gene-silencing by the tRNA maturase tRNase ZL under the direction of small guide RNA.</a> Gene Ther. 14, 78-85 (2007).</li><li>Elbarbary, R. A., Takaku, H., Tamura, M., Nashimoto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+vascular+endothelial+growth+factor+expression+by+TRUE+gene+silencing.">Inhibition of vascular endothelial growth factor expression by TRUE gene silencing.</a> Biochem. Biophys. Res. Commun. 379, 924-927 (2009).</li><li>Sano, T., Takahashi, M., Nozaki, T., Takahashi, Y., Tamura, M., Nashimoto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expanding+the+utility+of+heptamer-type+sgRNA+for+TRUE+gene+silencing.">Expanding the utility of heptamer-type sgRNA for TRUE gene silencing.</a> Biochem. Biophys. Res. Commun. 416, 427-432 (2011).</li><li>Iizuka, S., Oridate, N., Nashimoto, M., Fukuda, S., Tamura, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+inhibition+of+head+and+neck+squamous+cell+carcinoma+cells+by+sgRNA+targeting+the+cyclin+D1+mRNA+based+on+TRUE+gene+silencing.">Growth inhibition of head and neck squamous cell carcinoma cells by sgRNA targeting the cyclin D1 mRNA based on TRUE gene silencing.</a> PLoS One. 9 (114121), (2014).</li><li>Takahashi, M., 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=Elimination+of+specific+miRNAs+by+naked+14-nt+sgRNAs.">Elimination of specific miRNAs by naked 14-nt sgRNAs.</a> PLoS One. 7 (38496), (2012).</li><li>Takahashi, M., 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+naked+RNA+heptamer+targeting+the+human+Bcl-2+mRNA+induces+apoptosis+of+HL60+leukemia+cells.">A naked RNA heptamer targeting the human Bcl-2 mRNA induces apoptosis of HL60 leukemia cells.</a> Cancer Lett. 328, 362-368 (2013).</li><li>Watanabe, N., 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=Induction+of+apoptosis+of+leukemic+cells+by+TRUE+gene+silencing+using+small+guide+RNAs+targeting+the+WT1+mRNA.">Induction of apoptosis of leukemic cells by TRUE gene silencing using small guide RNAs targeting the WT1 mRNA.</a> Leukemia Res. 37, 580-585 (2013).</li><li>Elbarbary, R. A., 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=Modulation+of+gene+expression+by+human+cytosolic+tRNase+ZL+through+5&#8242;-half-tRNA.">Modulation of gene expression by human cytosolic tRNase ZL through 5&#8242;-half-tRNA.</a> PLoS One. 4, 5908 (2009).</li><li>Elbarbary, R. A., 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=Human+cytosolic+tRNase+ZL+can+downregulate+gene+expression+through+miRNA.">Human cytosolic tRNase ZL can downregulate gene expression through miRNA.</a> FEBS Lett. 583, 3241-3246 (2009).</li><li>Ninomiya, S., 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=Potential+small+guide+RNAs+for+tRNase+ZL+from+human+plasma,+peripheral+blood+mononuclear+cells,+and+cultured+cell+lines.">Potential small guide RNAs for tRNase ZL from human plasma, peripheral blood mononuclear cells, and cultured cell lines.</a> PLoS One. 10, 0118631 (2015).</li><li>Takahashi, M., 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=Screening+of+a+heptamer-type+sgRNA+library+for+potential+therapeutic+agents+against+hematological+malignancies.">Screening of a heptamer-type sgRNA library for potential therapeutic agents against hematological malignancies.</a> Leukemia Res. 38, 808-815 (2014).</li><li>Shivarov, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRUE+gene+silencing+for+hematologic+malignancies.">TRUE gene silencing for hematologic malignancies.</a> Leukemia Res. 38, 729 (2014).</li></ol>

In most cases, fully 2&#8242;-O-methylated, 5&#8242;- and 3&#8242;-phosphorylated heptamer-type sgRNAs dissolved in water-for-injection-grade water (in the concentration of 100 &#181;M) appear to be stable at below -20 &#176;C for at least one year, judging from their activity to induce apoptosis in cancer cells. However, their stability would change depending on their sequence, quality, and purity. In some cases, 5&#8242;- or 3&#8242;-phosphate of a part of sgRNA molecules appears to be removed spontaneously du...

真基因沉默（TR核苷酸酶Ž 信用证后称为- U tilizingËfficacious基因沉默）是RNA指导的基因沉默技术1中的一个。这种技术已经开发了基于tRNasežL（或3&#39;tRNase），tRNA的3&#39;加工核酸内切酶的特性：它可以通过识别切割的人造小导RNA（因组）的指导下，在任何预期的网站上的任何靶RNA 9 -靶RNA和因组2之间形成酰tRNA样前或微预tRNA的状结构。因组，这通常是7-31个核苷酸长，被分类为四组，5&#39;-半酰tRNA，七聚体RNA，14-nt的线性RNA和钩的RNA。 15 -我们已经通过引入各种人为设计sgRNAs入活细胞10展示为TRUE基因沉默的效果。我们还表明，sgRNAs可以被细胞的Wi被吸收thout任何转染试剂，可以下调它们的靶RNA水平和/或在人类癌细胞16诱导细胞凋亡- 18。 TRUE基因沉默似乎就通过tRNasežL和自然sgRNAs 19细胞内和细胞间的基因调控网络系统的工作- 21。 我们已经构建了含156七聚物型sgRNAs一个因组文库以寻找潜在的治疗七聚物型sgRNAs对于血癌22。我们筛选其对人骨髓瘤和白血病细胞的生存力的影响，并发现其中20能有效地诱导凋亡的肿瘤细胞株中的至少一种。和他们的4已经显示减少在小鼠异种移植实验肿瘤的生长速率。 在这里，我们提出了一个协议为七聚物型因组库潜在的治疗药物抗血癌的筛查。该协议包括如何构造因组文库，如何评估细胞生存力，以及如何进一步通过流式细胞术评估的有效sgRNAs每因组的效果。可以根据常规方法17,22虽然这些未在此方案包括进行分析因组的细胞靶RNA和有效sgRNAs评价在小鼠异种移植模型。

<table border="0" cellpadding="0" cellspacing="0" width="948">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">Custom heptamer RNA</td>
			<td style="width:167px;">Nippon Bioservice</td>
			<td style="width:144px;"></td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">OligoSep Prep HC Cartridge</td>
			<td style="width:167px;">Transgenomic</td>
			<td style="width:144px;">99-3860</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Water-for-injection-grade Water</td>
			<td style="width:167px;">Otsuka Pharmaceutical&#160;</td>
			<td style="width:144px;">7131400A2129</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI-1640 medium</td>
			<td style="width:167px;">Wako</td>
			<td style="width:144px;">189-02025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum</td>
			<td style="width:167px;">SIGMA-ALDRICH</td>
			<td style="width:144px;">172012-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">Penicillin-Streptomycin Mixed Solution</td>
			<td style="width:167px;">Nacalai Tesque</td>
			<td style="width:144px;">26253-84</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96 Well Plate</td>
			<td style="width:167px;">Greiner Bio-one</td>
			<td style="width:144px;">655 180</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell Counting Kit-8</td>
			<td style="width:167px;">DOJINDO</td>
			<td style="width:144px;">343-07623</td>
			<td>WST-8 solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microplate Reader, Sunrise Thermo RC-R</td>
			<td style="width:167px;">TEKAN</td>
			<td style="width:144px;">510-82851</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FACS Round-Bottom Tube</td>
			<td style="width:167px;">BD Falcon</td>
			<td style="width:144px;">60819-820</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate Buffered Saline</td>
			<td style="width:167px;">SIGMA-ALDRICH</td>
			<td style="width:144px;">P7059-1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PE Annexin V Binding Buffer</td>
			<td style="width:167px;">BD Biosciences</td>
			<td style="width:144px;">51-66121E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PE Annexin V</td>
			<td style="width:167px;">BD Biosciences</td>
			<td style="width:144px;">51-65875X</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">7AAD</td>
			<td style="width:167px;">SIGMA-ALDRICH</td>
			<td style="width:144px;">A9400-1MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flow Cytometer, FACSCalibur</td>
			<td style="width:167px;">BD Biosciences</td>
			<td style="width:144px;">342973</td>
			<td></td>
		</tr>
	</tbody>
</table>

true gene silencing: screening of a heptamer-type small guide rna library for potential cancer therapeutic agents

真基因沉默（TR核苷酸酶Ž 信用证后称为- U tilizingËfficacious基因沉默）是RNA指导的基因沉默技术，它利用的人造小导的RNA（因组）引导的tRNA 3&#39;加工内切核糖核酸酶，tRNasežL的一个，以识别靶RNA。 sgRNAs可以采取由细胞没有任何转染试剂，可以下调它们的靶RNA水平和/或在人类癌细胞中诱导细胞凋亡。我们筛选含有156对人骨髓瘤和白血病细胞的生存力的影响七聚物型sgRNAs一个因组文库，并发现其中20能有效地诱导凋亡的肿瘤细胞株中的至少一种。在这里，我们提出了一个协议为七聚物型因组库潜在的治疗药物抗血癌的筛查。该协议包括如何构造因组文库，如何评估细胞生存力，以及各因组的效果如何复rther评估通过流式细胞仪的有效sgRNAs。约2,000命中预计将通过筛选的16,384七聚体构成的全规模因组文库来获得。

六个七聚物型sgRNAs 5&#39;-AUCUUCA-3&#39;（H1885），5&#39;-ACACACA-3&#39;（H3277），5&#39;-GGGGGCG-3&#39;（H10927），5&#39;-GGGGCCC-3&#39;（H10944）， 5&#39;-GCCCCCG-3&#39;（H12287），和5&#39;-CACCAGC-3&#39;（H13260）作为含有5&#39;-和3&#39;-磷酸酯2&#39;-O-甲基RNA的化学合成。 这些sgRNAs检查用于在人白血病细胞系，HL60和人骨髓瘤细胞系，RPMI-8226的存活力的?...

Watch this Scientific Journal Video about TRUE Gene Silencing: Screening of a Heptamer-type Small Guide RNA Library for Potential Cancer Therapeutic Agents at JoVE.com

TRUE Gene Silencing: Screening of a Heptamer-type Small Guide RNA Library for Potential Cancer Therapeutic Agents

在这里，我们提出了一个协议为七聚物型因组库潜在的治疗药物抗血癌的筛查。

TRUE基因沉默：一个七聚物型小导RNA库的筛选潜在的癌症治疗药

TRUE gene silencing (termed after tRNase ZL-utilizing efficacious gene silencing) is one of the RNA-directed gene silencing technologies, which utilizes ...

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