This work was supported by Adaptable and Seamless Technology Transfer Program through Target-driven R&#38;D, Japan Science and Technology Agency, the Science Research Promotion Fund from the Promotion and Mutual Aid Corporation for Private Schools of Japan, and the JSPS KAKENHI Grant Numbers 24300342 and 15H04313.

1. Construction of a Heptamer-type sgRNA Library
<ol>
	<li>Select a subset of the 16,384 (47) 7-nt sequences unless the full-scale library is to be constructed. 
	NOTE: The sequences can be random and/or designed to target specific cellular RNAs. For the latter, find hairpin structures resembling the T-arm of tRNA in a target RNA with the aid of an appropriate computer program and/or visually, and select sequences complementary to 7-nt sequences immediately downstream of the hairpin structures4,10,...

<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>

Niigata University of Pharmacy and Applied Life Sciences has a pending patent application on a subset of the heptamer-type sgRNAs.

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...

TRUE gene silencing (termed after tRNase ZL-utilizing efficacious gene silencing) is one of the RNA-directed gene silencing technologies1. This technology has been developed based on properties of tRNase ZL (or 3&#8242; tRNase), tRNA 3&#8242; processing endoribonuclease: it can cleave any target RNA at any expected site under the direction of an artificial small guide RNA (sgRNA) by recognizing a pre-tRNA-like or micro-pre-tRNA-like structure formed between the target RNA and the sgRNA2-9. sgRNA, which is usually 7-31 nt in length, is categorized into four groups, 5&#8242;-half-tRNA, heptamer RNA, 14-nt linear RNA, and hook RNA.
We have demonstrated the efficacy of TRUE gene silencing by introducing various artificially-designed sgRNAs into living cells10-15. We have also shown that sgRNAs can be taken up by cells without any transfection reagents and can downregulate their target RNA levels and/or induce apoptosis in human cancer cells16-18. TRUE gene silencing appears to work on the intracellular and intercellular gene regulatory network system via tRNase ZL and natural sgRNAs19-21.
We have constructed an sgRNA library containing 156 heptamer-type sgRNAs to search for potential therapeutic heptamer-type sgRNAs for blood cancers22. We have screened it for the effect on viability of human myeloma and leukemia cells, and found that 20 of them can efficiently induce apoptosis in at least one of the cancer cell lines. And 4 of them have been shown to reduce tumor growth rates in mouse xenograft experiments.
Here we present a protocol for screening of a heptamer-type sgRNA library for potential therapeutic drugs against blood cancers. The protocol includes how to construct the sgRNA library, how to assess the effect of each sgRNA on cell viability, and how to further evaluate the effective sgRNAs by flow cytometry. Analysis for sgRNA&#39;s cellular target RNAs and evaluation of effective sgRNAs in mouse xenograft models can be performed according to conventional methods17,22, although these are not included in this protocol.

<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

TRUE gene silencing (termed after tRNase ZL-utilizing efficacious gene silencing) is one of the RNA-directed gene silencing technologies, which utilizes an artificial small guide RNA (sgRNA) to guide tRNA 3&#8242; processing endoribonuclease, tRNase ZL, to recognize a target RNA. sgRNAs can be taken up by cells without any transfection reagents and can downregulate their target RNA levels and/or induce apoptosis in human cancer cells. We have screened an sgRNA library containing 156 heptamer-type sgRNAs for the effect on viability of human myeloma and leukemia cells, and found that 20 of them can efficiently induce apoptosis in at least one of the cancer cell lines. Here we present a protocol for screening of a heptamer-type sgRNA library for potential therapeutic drugs against blood cancers. The protocol includes how to construct the sgRNA library, how to assess the effect of each sgRNA on cell viability, and how to further evaluate the effective sgRNAs by flow cytometry. Around 2,000 hits would be expected to be obtained by screening the full-scale sgRNA library composed of 16,384 heptamers.

The six heptamer-type sgRNAs 5&#8242;-AUCUUCA-3&#8242; (H1885), 5&#8242;-ACACACA-3&#8242; (H3277), 5&#8242;-GGGGGCG-3&#8242; (H10927), 5&#8242;-GGGGCCC-3&#8242; (H10944), 5&#8242;-GCCCCCG-3&#8242; (H12287), and 5&#8242;-CACCAGC-3&#8242; (H13260) were chemically synthesized as 2&#8242;-O-methyl RNA containing 5&#8242;- and 3&#8242;-phosphates.
These sgRNAs were examined for the effects on viability of a human leukemia ce...

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

Here we present a protocol for screening of a heptamer-type sgRNA library for potential therapeutic drugs against blood cancers.

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

The overall goal of this screening is to find potential therapeutic drugs against blood cancers.Begin this procedure by selecting a subset of Heptamer sequences.The sequences can be random, or designed to target specific cellular RNAs.To target specific cellular RNAs, use an appropriate computer program to find hairpin structures resembling the T-arm in a target RNA.Select complementary seven-nucleotide sequences immediately downstream of the hairpin.Using the phosphoramidite method on the controlled pore glass, or CPG support, synthesize each of the selected Heptamers as a fully 2-prime O-methylated, 5-prime and 3-prime phosphorylated RNA with the DNA-RNA synthesizer.Leave the 5-prime terminal DMT protecting group on in the last cycle of the synthesis.After completion of the last cycle, transfer the CPG with the synthesized oligonucleotide from the column to a glass vial with a teflon-lined screw cap.Add 1mL of 29%ammonium hydroxide and incubate the tube at 55

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5.6K vues.  Niigata University of Pharmacy and Applied Life Sciences. The overall goal of this screening is to find potential therapeutic drugs against blood cancers.Begin this procedure by selecting a subset of Heptamer sequences.The sequences can be random, or designed to target specific cellular RNAs.To target specific cellular RNAs, use an appropriate computer program to find hairpin structures resembling the T-arm in a target RNA.Select complementary seven-nucleotide sequences immediately downstream of the hairpin.Using the phosphoramidite method on the co...