sirna Synthesis Service mirna Synthesis Service In vivo sirna Custom Service: SAMiRNA sirna & mirna Phone: (ext.4->1)

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1 sirna Synthesis Service mirna Synthesis Service In vivo sirna Custom Service: SAMiRNA sirna & mirna Phone: (ext.4->1) SAMiRNA Phone:

2 01 sirna Synthesis Service AccuTarget TM sirnas 3 Genome-wide Predesigned sirna, Primer & Control AccuTarget Genome-wide Predesigned sirna Library 4 AccuTarget Real-Time PCR Primer Library 5 AccuTarget Control sirnas 6 Premade sirna & Primer AccuTarget Premade sirna Sets 10 AccuTarget Real-Time PCR Primer for Premade sirna Sets 12 Custom sirna AccuTarget Custom Designed sirna Synthesis 13 sirna Oligonucleotide FAQs 15 Users Protocol 16

3 AccuTarget TM sirnas Overview Small interfering RNA (sirna) 는 target specific gene silencing 을위한가장강력한수단으로유전자기능의확인, drug target 의 validation, 각종질병의 gene therapy 등에사용되고있습니다. sirna 는기존 antisense technology 에비해다음과같은장점을가지고있습니다. - 첫째, 적은수의 screening 으로도높은효율의 sirna 를찾을수있어시간과경비를절감할수있습니다. - 둘째, 낮은농도로효율적인 gene silencing 이가능합니다. - 셋째, natural biological mechanism 으로 target specificity 가매우높습니다. sirna mechanism sirna 는약 21-nucleiotide sense/anti-sense RNA 로구성된 double-strand RNA 입니다. sirna 는세포내에서 RNA-induced silencing complex (RISC) 라고불리는 protein complex 와결합합니다. Single strand RNA 가 RISC 에의해선택및장착되어 ribonucleoprotein complex 를형성하고, 이러한과정을통하여활성화된 RISC 의 single strand RNA 는상보적인서열의 target mrna 에결합하여이를분해합니다. Turbo si-designer - Bioneer s proprietary sirna design algorithm sirna 는최근새로운 functional genomics 의 tool 로서각광을받고있고, 그중요성을인정받아 2002 년도 Science 저널에서 small RNA (sirna 와 mirna) 가 Breakthrough of the year 1 번으로선정되었습니다. RNAi 연구에있어서가장중요한요소가운데하나는 효율이높은 target site 를찾는것 입니다. Target site 마다이에상응하는 sirna 의효율이다르기때문에, 고효율의 sirna target site 를찾는것이정확한실험결과를얻는데매우중요합니다. 바이오니아에서는 2003 년 10 월부터산업자원부지원하에 sirna design algorithm 개발프로젝트를시작하였고주관기관인바이오니아와위탁기관인국가유전체정보센터 (NGIC, 책임자 ; 김영주박사 ) 가공동으로 3 년에걸쳐총 9 억원을투입하여효능이뛰어난 sirna design algorithm 을개발하였습니다. 본과제의주목적은기존의연구를통해이미알려져있는 nonfunctional 한 sirna site 를배제시키고다른여러변수들을고려하여효율적인 sirna target site 를찾는 algorithm 을개발하는것이었습니다. 이렇게일차적으로개발된 algorithm 을이용하여예측한 sirna target site 의효율확인을위한실험을진행하고, 각변수가실험결과에미치는영향을분석하여보다효율적인 sirna target site 를높은확률로예측할수있는 design program 인 Turbo si-designer 를완성했습니다. Turbo si-designer 의성능을확인하기위하여항세포사멸및세포분열관련유전자인 survivin 을 target 으로 82 개의 sirna 를 design 하여 A549 lung carcinoma 세포에 transfection 후, northern blot 과 QuantiGene kit 로 mrna 의발현량을측정하였습니다. 점수가낮은 sirna (Figure 1B) 는점수가높은 sirna (Figure. 1A) 에비해효율이좋지않음을확인하였고, 이를통해 Turbo si-designer 가비효율적인 sirna site 들을매우효과적으로배제하여고효율의 sirna 를찾을확률을높여주는것을알수있습니다 (Figure1). Figure 1. sirna knockdown efficiency of sirnas designed by Turbo si- Designer was analyzed by Northern blot and Real-Time PCR analysis. A) sirna knockdown efficiency of high NGIC score sirnas. B) sirna knockdown efficiency of low NGIC score sirnas. 3

4 AccuTarget Genome-wide Predesigned sirna Library Description AccuTarget TM Genome-wide Predesigned sirna Library 는한국생명공학연구원국가유전체정보센터와바이오니아가공동개발한 sirna design algorithm인 Turbo si-designer를이용하여 predesign된 sirna입니다. 이를사용하여 knockdown 효율이우수한 AccuTarget TM Genomewide Predesigned sirna database를구축하였습니다. Human 18,048 개, mouse 17,117개, rat 8,392개유전자에대한 sirna가 design되어있어주문접수후바로합성하여배송가능합니다. The Bioneer Guarantee 동일유전자에대한 3개의 predesigned sirna를구입하시면, 이중 2개가 target mrna expression level을 80% 이상억제함을보장합니다. Target 유전자의 mrna 수준에서 80% 이상감소가없을경우, 무료로 2개의 sirna를제공합니다. * 단, 본사에서요하는다음과같은 supporting data를별도로제출해야합니다. 1. sirna knockdown efficiency data: NC (AccuTarget TM Negative Control) and sirna concentration at 100 nm 2. Transfection efficiency data: PC (AccuTarget TM GAPDH/GFP/ Luciferase sirna) and NC (AccuTarget TM Fluorescein-labeled Negative Control) Application AccuTarget TM Genome-wide Predesigned sirna Library는다양한 RNAi 실험에활용할수있습니다. Functional genomics and proteomics research Gene expression studies Array analysis Ordering Information Purification Bio-RP Guaranteed nmole 10 nmole 20 nmole 50 nmole 100 nmole 10 nmole 20 nmole 50 nmole 100 nmole Features and Benefits High sirna knockdown rates: 동일유전자에대한 3개의 sirna 중 2개는 target mrna expression level을 80% 이상억제합니다 (Figure 2). Unique design algorithm: Off target effect를최소화하면서 sirna knockdown 효율을최대화합니다. Competitive pricing: 경제적인비용으로극대화된효율을경험하세요. Figure 2. sirna knockdown efficiency of AccuTarget TM Genome-wide Predesigned sirna. AccuTarget TM Predesigned sirnas are highly effective. To determine sirna knockdown efficiency of predesigned sirnas, HeLa cells were transfected with sirnas at 100 nm concentration. 24 hr post-transfection, total RNA was isolated and the level of target mrna was measured by qrt-pcr. This data demonstrates the effectiveness of the Turbo si-designer algorithm: 83.8% of tested sirnas induced >70% sirna knockdown and 38.1% of tested sirnas elicited >90% knockdown. BIONEER 4

5 AccuTarget Real-Time PCR Primer Library Description 바이오니아는 sirna knockdown validation에필요한 Real-Time PCR Primer Library를제공합니다. Human predesigned sirna에대해실험적으로검증된 11,154개의 Real-Time PCR primer set을구비하고있습니다. 바이오니아홈페이지에서 predesigned sirna를검색하시면해당 Real-Time PCR primer가함께검색되어 primer를 design 하고검증하는시간을절약할수있습니다. 모든 primer의증폭효율은자사 Real-Time PCR 기기인 Exicycler TM 96과 AccuPower GreenStar TM qpcr PreMix를이용하여검증하였습니다 (Figure 3). Features and Benefits In ready-to-ship format: 11,154개 human 유전자특이적 primer All primers pre-validated: MALDI-TOF mass spectrometer를통한 QC검증 Competitive pricing: 경제적인비용으로극대화된효율을경험하세요. Figure 3. Real-Time PCR validation test of human oxidoreductase using AccuTarget TM Human Oxidoreductase Real-Time PCR Primer set. Ordering Information AccuTarget TM Real-Time PCR Primer for Individual Gene Cat. no. Product Description Reactions PHS-P01 AccuTarget TM Human Real-Time PCR Primer for Individual Gene 100 rxns PHS-P02 AccuTarget TM Human Real-Time PCR Primer for Individual Gene 200 rxns 5

6 AccuTarget Control sirnas Description AccuTarget TM Positive Control sirna는 target gene에대하여높은효율의 knockdown 효과를나타냅니다 (Figure 4, 5 & 6). Housekeeping gene으로널리사용되는 GAPDH 및 reporter system으로잘알려진 GFP와 luciferase에대한 positive control sirna를제공합니다. 또한 mouse positive control로는 Lamin A/C or PPIB (cyclophilin B) 에대해효율적인 sirna를제공합니다. Human과 mouse, rat에서공통으로사용가능한 AccuTarget TM Negative Control sirna는 non-targeting sirna로써, human과 mouse, rat의알려진모든유전자들에대해 homology가낮은 sequence입니다. 따라서모든유전자의 knockdown 실험에 negative control로사용할수있어서편리합니다. Transfection 효율을쉽게관찰하기위하여 sirna uptake를확인할수있는 fluoresceinlabeled Negative Control sirna도함께제공합니다 (Figure 7). 또한간편하고저렴한 positive와 negative control sirna로구성된 control set도제공합니다. AccuTarget TM Positive Control sirna와 Negative Control sirna는바이오니아홈페이지에서원하는 scale 과 purification을선택하여주문이가능합니다. Features and Benefits Excellent performance: 90% 이상의 knockdown 효율을갖는 positive control sirna Monitoring of transfection rate: 형광물질로 labeling되어편리하게효능검증이가능한 negative control sets Competitive pricing: 경제적인비용으로극대화된효율을경험하세요. Positive Control sirna 1. GAPDH-siRNA A) Northern blotting B) qrt-pcr Figure 4. Effects of Human GAPDH Positive Control sirna. HeLa cells were transfected separated with AccuTarget TM Human GAPDH Positive Control and Negative Control sirna using lipofectamine 2000 (Invitrogen) at a final concentration of 100 nm. Total cellular RNA was isolated from transfected cells 24 hr after transfection and subjected to Northern blot and Real-Time PCR analysis. As can be seen from Figure 4-B, about 3% GAPDH mrna remained. 2. GFP-siRNA BIONEER Figure 5. HeLa cells in a 24-well plate were co-transfected with 200 ng of CMV-GFP plasmid and 10 nm of GFP sirna using lipofectamine 2000 transfection reagent. Next day, the expression of GFP was observed by using a Nikon Eclipse TS100 epifluorescence microscope. In contrast to bright green fluorescence of GFP protein in NC-siRNA-transfected cells, no fluorescence was detected from GFP-siRNA-transfected cells, indicating efficient knockdown of GFP by using our positive control GFP-siRNA. 6

7 AccuTarget Control sirnas 3. Luciferase-siRNA Figure 6. HeLa cells in a 6-well plate were co-transfected with 400 ng of CMV-luc plasmid and 10 nm of luciferase sirna using lipofectamine 2000 transfection reagent. Next day, cells were harvested and assayed for luciferase activity. As shown in Figure. 6, cotransfection with our positive control luciferase sirna led to efficient knockdown of luciferase activity (85% - 95% knockdown compared to luciferase activity of NC-siRNA-transfected cells). 4. Mouse Positive Control sirna Figure 7. Effects of Mouse Lamin A/C Positive Control sirna. Hepa1-6 cells were transfected separately with AccuTarget TM Mouse Lamin A/C Positive Control and Negative Control sirna using Lipofectamine RNAimax (Invitrogen) at a final concentration of 1 nm. Total cellular RNA was isolated from transfected cells 24 hr after transfection and subjected to Real-Time PCR analysis. Figure 8. Effects of Mouse PPib (Cyclophilin B) Positive Control sirna. Hepa1-6 & NIH 3T3 cells were transfected separately with AccuTarget TM Mouse PPib (Cyclophilin B) Positive Control and Negative Control sirna using Lipofectamine RNAimax (Invitrogen) at a final concentration of 5 & 1 nm. Total cellular RNA was isolated from transfected cells 24 hr after transfection and subjected to Real-Time PCR analysis. 7

8 AccuTarget Control sirnas Negative Control sirna Figure 9. HeLa cells transfected with FITC-labeled sirna (Cat. no. SN-1021) was observed by confocal microscopy. The fluorescent cells indicate that the target cells were successfully transfected with the sirna. Ordering Information AccuTarget TM Positive Control sirnas BIONEER Cat. no. Description Purification Guaranteed Yield SP-1001 AccuTarget TM GAPDH Positive Control sirna Bio-RP 5 nmole SP-1002 AccuTarget TM GAPDH Positive Control sirna Bio-RP 10 nmole SP-1003 AccuTarget TM GAPDH Positive Control sirna Bio-RP 20 nmole SP-1011 AccuTarget TM GAPDH Positive Control sirna 5 nmole SP-1012 AccuTarget TM GAPDH Positive Control sirna 10 nmole SP-1013 AccuTarget TM GAPDH Positive Control sirna 20 nmole SP-2001 AccuTarget TM GFP Positive Control sirna Bio-RP 5 nmole SP-2002 AccuTarget TM GFP Positive Control sirna Bio-RP 10 nmole SP-2003 AccuTarget TM GFP Positive Control sirna Bio-RP 20 nmole SP-2011 AccuTarget TM GFP Positive Control sirna 5 nmole SP-2012 AccuTarget TM GFP Positive Control sirna 10 nmole SP-2013 AccuTarget TM GFP Positive Control sirna 20 nmole SP-3001 AccuTarget TM Luciferase Positive Control sirna Bio-RP 5 nmole SP-3002 AccuTarget TM Luciferase Positive Control sirna Bio-RP 10 nmole SP-3003 AccuTarget TM Luciferase Positive Control sirna Bio-RP 20 nmole SP-3011 AccuTarget TM Luciferase Positive Control sirna 5 nmole SP-3012 AccuTarget TM Luciferase Positive Control sirna 10 nmole SP-3013 AccuTarget TM Luciferase Positive Control sirna 20 nmole SP-4001 AccuTarget TM Mouse Lamin A/C Positive Control Bio-RP 5 nmole SP-4002 AccuTarget TM Mouse Lamin A/C Positive Control Bio-RP 10 nmole SP-4003 AccuTarget TM Mouse Lamin A/C Positive Control Bio-RP 20 nmole SP-4011 AccuTarget TM Mouse Lamin A/C Positive Control 5 nmole SP-4012 AccuTarget TM Mouse Lamin A/C Positive Control 10 nmole SP-4013 AccuTarget TM Mouse Lamin A/C Positive Control 20 nmole SP-5001 AccuTarget TM Mouse cyclophilin B c Positive Control Bio-RP 5 nmole SP-5002 AccuTarget TM Mouse cyclophilin B c Positive Control Bio-RP 10 nmole SP-5003 AccuTarget TM Mouse cyclophilin B c Positive Control Bio-RP 20 nmole SP-5011 AccuTarget TM Mouse cyclophilin B c Positive Control 5 nmole SP-5012 AccuTarget TM Mouse cyclophilin B c Positive Control 10 nmole SP-5013 AccuTarget TM Mouse cyclophilin B c Positive Control 20 nmole 8

9 AccuTarget Control sirnas AccuTarget TM Negative Control sirnas Cat. no. Description Purification Guaranteed Yield SN-1001 AccuTarget TM Negative Control sirna Bio-RP 5 nmole SN-1002 AccuTarget TM Negative Control sirna Bio-RP 10 nmole SN-1003 AccuTarget TM Negative Control sirna Bio-RP 20 nmole SN-1011 AccuTarget TM Negative Control sirna 5 nmole SN-1012 AccuTarget TM Negative Control sirna 10 nmole SN-1013 AccuTarget TM Negative Control sirna 20 nmole SN-1021 AccuTarget TM Fluorescein-labeled Negative Control sirna 5 nmole SN-1022 AccuTarget TM Fluorescein-labeled Negative Control sirna 10 nmole SN-1023 AccuTarget TM Fluorescein-labeled Negative Control sirna 20 nmole AccuTarget TM Control sirna Sets Cat. no. Description Purification Guaranteed Yield SS-1001 AccuTarget TM GAPDH Control sirna Set Bio-RP SS-1002 AccuTarget TM GFP Control sirna Set Bio-RP SS-1003 AccuTarget TM Luciferase Control sirna Set Bio-RP SS-1011 AccuTarget TM GAPDH Control sirna Set SS-1012 AccuTarget TM GFP Control sirna Set SS-1013 AccuTarget TM Luciferase Control sirna Set 5 nmole positive control + 2 nmole negative control 5 nmole positive control + 2 nmole negative control 5 nmole positive control + 2 nmole negative control 5 nmole positive control + 2 nmole negative control 5 nmole positive control + 2 nmole negative control 5 nmole positive control + 2 nmole negative control 9

10 AccuTarget Premade sirna Sets Description AccuTarget TM Premade Human sirna Sets은즉시 transfection 실험에사용할수있도록제작된 54,144종의 sirna로이루어진제품입니다. 특정 cellular process, 암, 질병등의연구목적에특화된 25개의 biological pathway ( 혹은 gene family) 별로제작된 sirna library set를 0.1, 0.25, 0.5, 1 nmole 로주문제공하며, 각 set에포함되어있는 sirna는개별로도 10, 20, 50, 100 nmole의다양한용량으로주문가능합니다. Premade sirna의 validation 과정은다음과같습니다. Features and Benefits Categorized by pathway / family: 연구수행에편리하게특화된 format Pre-validated sirna libraries available: 즉시사용가능한검증된효능의 sirna library Competitive pricing: 경제적비용으로극대화된효율을경험하세요. Application AccuTarget TM Premade sirna Set은다양한 RNAi 실험에활용할수있습니다. Pathway analysis and target identification and validation Drug target HTS (High Throughput sirna Screening) BIONEER 10

11 AccuTarget Premade sirna Sets Figure 10. Knockdown efficiency of AccuTarget TM sirna Library. To determine knockdown efficiency, HeLa cells were transfected with individual sirnas at 100 nm concentration. 24 hr post-transfection, total RNA was isolated and the level of target mrna was measured by qrt-pcr. This data demonstrates the effectiveness of the Turbo si-designer algorithm: 83.8% of tested sirnas induced >70% knockdown and 38.1% of tested sirnas elicited >90% knockdown. Figure 11. sirna knockdown efficiency of AccuTarget TM Human Validated Cell Cycle sirna Set. HeLa cells were transfected with sirnas targeting 33 different genes at a concentration of 20 nm. Total RNA was isolated and the level of target mrna was measured by qrt-pcr. This data demonstrates the effectiveness of the Turbo si-designer algorithm. Ordering Information Cat. no. Product Name AccuTarget TM Premade Human sirna Sets No. of Genes Cat. no. Product Name SHS-001 Antioxidant sirna set 38 SHS-013 Lyase sirna set 123 SHS-002 Apoptosis sirna set 290 SHS-014 Motor sirna set 122 SHS-025 Cancer sirna set 1157 SHS-015 NF-kB pathway sirna set 37 SHS-003 Caspase sirna set 37 SHS-016 Nucleic acid binding sirna set 2573 SHS-004 Cell cycle sirna set 112 SHS-017 Oxidoreductase sirna set 551 SHS-005 Cyclase sirna set 21 SHS-018 Peptidase sirna set 491 SHS-006 Cytochrome P450 sirna set 52 SHS-019 Phosphatase sirna set 188 SHS-007 Deaminase sirna set 22 SHS-020 Receptor sirna set 1516 SHS-008 GPCR signaling pathway sirna set 727 SHS-021 Transferase sirna set 1428 SHS-009 Helicase sirna set 114 SHS-022 Transporter sirna set 1021 SHS-010 Isomerase sirna set 104 SHS-023 Tubulin sirna set 20 SHS-011 Kinase sirna set 699 SHS-024 Ubiquitin sirna set 77 SHS-012 Ligase sirna set 272 No. of Genes 11

12 AccuTarget Real-Time PCR Primer for Premade sirna Sets Description Premade sirna set 에대한 Real-Time PCR Primer Library 는유전자기능별 pathway 분류에따른 10,635 개의 primer set 로제공됩니다. SYBR Green (AccuPower GreenStar TM qpcr PreMix, Cat. no. K-6210) 을이용한 Real-Time PCR 에본제품을이용할경우더욱 specific 하고 sensitive 한결과를얻을수있습니다. Ordering Information Product Description no. of Genes Reactions / gene AccuTarget TM Human Antioxidant Real-Time PCR primer Set rxns AccuTarget TM Human Apoptosis Real-Time PCR primer Set rxns AccuTarget TM Human Cancer Real-Time PCR primer Set 1, rxns AccuTarget TM Human Caspase Real-Time PCR primer Set rxns AccuTarget TM Human Cell cycle Real-Time PCR primer Set rxns AccuTarget TM Human Cyclase Real-Time PCR primer Set rxns AccuTarget TM Human Cytochrome P450 Real-Time PCR primer Set rxns AccuTarget TM Human Deaminase Real-Time PCR primer Set rxns AccuTarget TM Human GPCR signaling pathway Real-Time PCR primer Set rxns AccuTarget TM Human Helicase Real-Time PCR primer Set rxns AccuTarget TM Human Isomerase Real-Time PCR primer Set rxns AccuTarget TM Human Kinase Real-Time PCR primer Set rxns AccuTarget TM Human Ligase Real-Time PCR primer Set rxns AccuTarget TM Human Lyase Real-Time PCR primer Set rxns AccuTarget TM Human Motor Real-Time PCR primer Set rxns AccuTarget TM Human NF-kB pathway Real-Time PCR primer Set rxns AccuTarget TM Human Nucleic acid binding Real-Time PCR primer Set 2, rxns AccuTarget TM Human Oxidoreductase Real-Time PCR primer Set rxns AccuTarget TM Human Peptidase Real-Time PCR primer Set rxns AccuTarget TM Human Phosphatase Real-Time PCR primer Set rxns AccuTarget TM Human Receptor Real-Time PCR primer Set 1, rxns AccuTarget TM Human Transferase Real-Time PCR primer Set 1, rxns AccuTarget TM Human Transporter Real-Time PCR primer Set rxns AccuTarget TM Human Tubulin Real-Time PCR primer Set rxns AccuTarget TM Human Ubiquitin Real-Time PCR primer Set rxns BIONEER 12

13 Custom Designed sirna Synthesis Description Custom sirna 는고객이요구하는 sirna sequence 를합성해드리는서비스입니다. 바이오니아에서 sirna 를합성하는경우에는 free-design 서비스를제공받을수있습니다. Pre-designed sirna 이외의유전자는 Turbo si-designer에의해제작및합성이가능하며, 하나의유전자에대하여세개이상의 sirna 를 design 및합성할수있습니다. 정제방법으로 Bio-RP, 정제를선택하실수있습니다. 또한원하시는 sirna sequence에다양한 modification도가능합니다. 30 mer의 sirna를최대 32 개의서로다른 3 overhang 선택을포함한다양한옵션을선택함으로써다양한형태의 sirna를주문할수있습니다. sirna는정제방법, annealing 유 / 무, dry 또는 ready-to-use 등의선택옵션에따라제작되어제공됩니다. Figure 1. MALDI-TOF mass spectrometry analysis of a custom sirna. All sirnas are subjected to MALDI-TOF mass spectrometry to ensure its quality. Modification 문의는 sirna-support@bioneer.co.kr로메일을보내주시거나 (042) 으로연락주시기바랍니다. Custom sirna 는본사의최첨단 clean room 시설에서합성된후바이오니아의 Bio-RP purification system 을통해정제됩니다. 정제된 sirna 는 MALDI-TOF mass spectrometry 를이용한품질검사를시행하여합격된 sirna 만을발송합니다. 보다높은순도의 sirna 를원하신다면, purification 선택을추천합니다 (Figure 1). Annealed sirna 를요청하신경우에는동량의 sense 와 antisense RNA 를 annealing 하여 double strand form 으로제작한다음, PAGE 로 double strand sirna 가제대로만들어졌는지확인한후제공해드립니다 (Figure 2). Features and Benefits Guaranteed performance: 자사 sirna design program을이용하여 custom sirna를 design 하실경우, 3개중 2개가적어도 80% 의 knockdown 효율을나타냅니다. 그렇지않을경우무료로 2개의 sirna를추가로제공합니다. High quality: 모든 sirna는 clean room에서제조되며 MALDI-TOF 및 PAGE 로검증됩니다. Free sirna design service: Turbo si-designer software를사용하여무료로 design 해드립니다. Competitive pricing: 무료로 overhang 및 annealing 서비스가제공되므로경제적비용으로제품을이용할수있습니다. Figure 2. PAGE data of annealed double-stranded custom sirna. Complementary single-strand RNA strands were hybridized to form sirna duplex and analyzed by 15% non-denaturing PAGE. SS: single-strand RNA, DS: double-strand sirna. Ordering Information Cat. no. Purification Guaranteed nmole S nmole S nmole Bio-RP S nmole S nmole S nmole S nmole S nmole S nmole Single strand 형태의 sense 및 antisense RNA 각각을동일한농도로 dry 하여제공해드립니다. Annealing buffer 와 DEPC D.W. 를기본으로제공해드립니다. Annealing 된상태의 sirna 를원하실경우는 2 만원의비용이추가됩니다. 모든합성 RNA 에대해 MALDI-TOF QC data 를첨부해드립니다. 13

14 Custom Designed sirna Synthesis Modification available for custom sirna 5'- Modification 3'- Modification Internal Modification Purification Guaranteed Yield 5' Fluorescein 3' Fluorescein Phosphorothioate 5' Phosphorylation 3' Phosphorylation 2'-OMe-rA, rc, rg, ru 5' Biotin 3' Biotin 2'-F(A) 5' Amine 3' Amine 2'-F(U) 10 nmole, 5' TAMRA 3' TAMRA 2'-F(G) 20 nmole 5' Thiol 3' Thiol 2'-F(C) 50 nmole 5' Cy3 3' DABCYL Inosine 100 nmole 5' Cy5 3' Cholesterol Deoxy-abase 5' PEG ' PEG 2000 Chimeric DNA 5 Cy5.5 5 Cy5.5 신속하고전문적인고객서비스 sirna 실험이익숙하지않은고객들도본사의제품을구입하시면언제든지실험관련상담을받으실수있습니다. sirna 를이용한다양한실험들에대해수년간의경험이축적된바이오니아의연구원들이고객들의질문에대해신속하고친절하게답해드립니다. 제품및실험관련문의사항은아래이메일또는전화번호로연락을주시면당일에성실히답변해드립니다. sirna 제품및실험관련상담 sirna@bioneer.co.kr, sirna-support@bioneer.co.kr Tel: ( 올리고팀 ) BIONEER 14

15 sirna Oligonucleotide FAQs 1. 주문해서받게되는 sirna 의상태는어떤가요? AccuTarget TM Genome-wide Predesigned sirna, Premade sirna Sets, Control sirna 는 sense 와 antisense 각각을동일농도로정제한다음 Annealing 과정을거쳐건조된상태의 duplex 로제공합니다. 이를고객이원하는 buffer, 또는, 함께동봉해드리는 DEPC D.W. 를이용하여원하는농도 (100 μm 로권장함 ) 로녹여서사용하시면됩니다. Custom sirna 주문시 annealing service 를신청하시면 sense 와 antisense 각각을동일농도로정제한다음 Annealing 과정을거쳐건조된상태의 duplex 로제공해드립니다. 이를고객이원하는 buffer 또는, 함께동봉해드리는 DEPC D.W. 를이용하여원하는농도 (50 μm 로권장함 ) 로녹여서사용하시면됩니다. Custom sirna 주문시 annealing service 를신청하지않으신경우 annealing buffer(1x) 를이용하여동봉되어있는 annealing protocol 대로진행하시면됩니다. 2. In vitro에서실험을하려고하는데어떤 scale의 sirna 를주문하는것이좋을까요? 그리고 purification은어떤방법을선택해야할까요? 10 nmole scale의 sirna를 100 nm로 transfection 한다면 6 well plate 기준으로 100개 well에 transfection 할수있는양입니다. In vivo 실험이아니라면 Bio-RP Purification을선택하셔도좋은결과를얻으실수있습니다. In vivo 실험에서는 purification을이용하시는것을권장합니다. 3. AccuTarget TM Genome-wide Predesigned sirna를주문하였는데효능이좋지않았습니다. 어떻게해야할까요? 먼저 sirna를 PAGE로확인해보시고 sirna가분해되지않았다면 transfection 효율을확인해보셔야합니다. 한유전자당 3개의 Predesigned sirna를구매하신후그중 2개가 80% 의 knockdown 효율을보이지않을경우, 2개의 sirna를무상으로제공해드립니다. 다만아래의 supported data 제출이필요합니다. 1. sirna 효율측정 data - NC : AccuTarget TM negative control, sirna 농도 : 100 nm 2. Transfection 효율측정 data - PC : AccuTarget TM GAPDH/GFP/Luciferase sirna - NC: AccuTarget TM Fluorescein labeled negative control 4. 합성된 sirna에는 5 또는 3 위치에 phosphate가붙어있나요? 주문하실때별도로선택하지않으면 5 과 3 위치에는 -OH 기가부착된상태로제공됩니다. 따라서 5 phosphate가부착된 sirna를원하시면 5 phosphorylation modification 된 sirna를주문하셔야합니다. 5. sirna는어떻게보관하며얼마나오래가나요? 일반적으로 sirna는 -20 에서 1년이상안정합니다. 특히건조한상태에서는매우안정적이며오래보관하실수있습니다. 용액상태에서도안정하지만 RNase 오염등으로인해 degradation 될수도있습니다. 또한 sirna는 얼렸다녹였다 를반복하게되면분해를촉진시켜품질이상이발생될수있으므로합성 sirna는받으신후에는필요량만큼여러개로분주하여각각을보관하신뒤하나씩꺼내어사용하시는것이바람직합니다. RNA의경우, 높은 ph에서는 hydrolysis에의해 phosphodiester bond가깨질수있으니주의를요합니다. 6. Fluorescent dye modified sirna는어떻게보관해야하나요? 빛에노출되면형광이약해질수있으므로빛이차단된장소에서차광용기에보관하시는것이좋습니다. Cy3, Cy5는 ph9 이상에서분해될수있으므로 Cy3, Cy5 modified sirna 보관시에는염기성환경을피해야합니다. 7. 합성된 sirna가몇 ng 인지알수있나요? 일반적으로 sirna의합성은 guarantee nmole 단위로신청되며합성된결과도보통 nmole 단위로 report 됩니다. ng 또는 μg 단위로사용하시려면 mole을 g으로환산하시면됩니다. Report에 sirna의분자량이표시되기때문에아래식을이용하여쉽게 g으로바꿀수있습니다. 분자량 M.W. (g) X mole 수 (nmole) = sirna의양 (ng) 8. sirna 실험을처음시작하려고합니다. 어떻게실험조건을잡을까요? sirna 실험에서중요한것은 sirna가 cell 내에제대로 delivery 되는가하는점입니다. 현재판매되고있는 positive control이나 NC-FITC를이용하여간단하게 sirna delivery를모니터링하실수있습니다. 9. sirna 실험을하기전에유념해야할점은무엇입니까? 첫째, sirna 염기서열이모두같은효율로유전자발현을억제하지않기때문에 3~4개의 sirna를시험하여가장적합한 sirna를찾아야합니다. 둘째, 단백질수준에서억제효과가 sirna 작용에의한것임을정확히확인하기위해서 mrna 수준에서의측정도이루어져야합니다. 마지막으로원하는 knockdown phenotype을확인하기위해한유전자당 2개의다른 sequence의 sirna로실험하여같은 phenotype이나타나는지확인해보는것이바람직합니다 nmole의 sirna로어떻게 100 nm로 transfection 할수있을까요? 가장많이혼동하시는부분입니다. sirna를 100 nm로 6 well plate의 1 well에서 transfection 하실때, transfection volume이 1 ml이므로 100 nm 농도 (100 pmole/ml) 로 sirna를처리하려면 100 pmole 의 sirna가필요합니다. 50 μm (50 pmole/μl) stock의 sirna를 2 μl 넣으면 100 pmole의 sirna를 transfection 한것이됩니다. 만약, 10 nmole 주문하시면 100 nm (100 pmole/ml) transfection 시 100개 well에사용하실수있습니다. 11. sirna의 transfection efficiency를확인하는방법은무엇인가요? NC-FITC를 transfection 후형광현미경관찰을통해 transfection efficiency 확인이가능합니다. 또한 NC-FITC는 sirna와 transfection reagent의사용농도및세포조건의확립등본실험에앞서실험조건을셋팅하기위한테스트시약으로사용될수있습니다. 12. sirna의 knockdown 효율확인을위한실험방법은무엇인가요? qrt-pcr, northern blot, western blot 등의실험을통해 knockdown 효율을확인하실수있습니다. 15

16 User Protocol Dilution Protocol 1. Briefly centrifuge tubes (or multi-well plates) containing sirna to ensure that the sirna pellet is located at the bottom of the tube. 2. Dissolve sirnas to a convenient stock concentration using the recommended volume of DEPC-D.W. (or RNase-free water) shown in Table Pipette the solution up and down 3~5 times (or vortex briefly). 4. Briefly centrifuge tubes (or multi-well plates) containing sirna to ensure that the solution is collected at the bottom of the tube. 5. Aliquot the sirnas into small volumes and store at -20. sirna is stable for 1 year under the specified storage condition. For best results, limit freeze-thaw events for each tube no more than five. Table 1. Recommended sirna resuspension volumes and concentrations. sirna Amount (nmol) DEPC-D.W. volume(μl) for desired final concentration 100 μm stock 20 μm stock Exceeds tube volume Transfection Protocol * We use the Lipofectamine TM RNAiMAX (Invitrogen; Cat.no ) and HeLa cell for transfection procedure. * This protocol is fixed at 6-well plate in vitro culture condition (if you want to change this condition, you have to consider the relative surface area (Table 2) and Invitrogen protocol, when you are seeding the cells into the culture dish). 1. One day (24 hr) before transfection, plate 3.0Ⅹ10 5 HeLa cells in each well with 2.5 ml of growth medium without antibiotics such that they will be 50~60% confluent at the time of transfection. 2. Remove the growth medium from the 6-well plate before transfection. And add the 500 μl fresh growth medium without serum in each well. 3. For each well to be transfected, prepare sirna duplex- Lipofectamine TM RNAiMAX complexes as follows Dilute sirna duplex (making final concentration as 5 nm~100 nm) in 250 μl growth medium (or Opti-MEM I Reduced Serum medium) without serum. Mix gently by vortex Mix Lipofectamine TM RNAiMAX gently before use, then dilute 3.5 μl in 250 μl medium (or Opti-MEM I Reduced Serum medium) without serum. Incubate this solution 5 minutes at room temperature Combine the diluted sirna duplex with the diluted Lipofectamine TM RNAiMAX. Mix and incubate for 20 minutes at room temperature. 4. Add the mixture to each well containing HeLa cells, which result 1 ml as total volume. Mix gently by hand rocking the plate back and forth. 5. Incubate the cells for 5~6 hr at 37 in CO2 incubator. 6. Change the medium with fresh one containing serum and incubate the cells 24~48 hr until you are ready to assay for gene knockdown. Table 2. The relative surface area of in vitro cell culture dish and culture media volume. Culture Vessel Relative surface area Volume of plating medium 96-well μl 48-well μl 24-well μl 6-well ml 60 mm 10 5 ml 100 mm ml BIONEER 16

17 02 mirna Synthesis Service AccuTarget TM mirnas 18 AccuTarget TM mirna mimics & inhibitors 19 AccuTarget TM mirna Controls 21

18 AccuTarget mirnas Overview MicroRNA (mirna) 는 21~25 nucleotide (nt) 의 small non-coding RNA 분자로서진핵생물의유전자발현을제어하는조절물질입니다. 사람에는약 1,700 여종의 mirna가알려져있으며 (mirbase version 21), 이러한 mirna는특정세포에서특정시점에발현되어 mrna의발현을정교하게조절합니다. 주로 mrna의 3 -UTR에결합하여발생, 분화, 증식, apoptosis, stress response 등을포함한대부분의세포기능조절에관여하는것으로알려져있습니다. mirna의생성은두단계의과정으로이루어집니다. 최초의 mirna 전사체 (primary mirna/pri-mirna) 가핵안에서 Drosha라는 RNaseIII type 효소에의해 70~90 nt 정도의 stem-loop 구조의 premirna로만들어지고, 이후세포질로이동하여 Dicer라는효소에의해절단되어 21~25 nt의 mature mirna가됩니다 (Figure 1). mirna는생명공학연구에새로운통찰력을제공하며, 비교적최근에발견된물질임에도불구하고, post-transcriptional level에서가장중요한유전자 regulator로인정되고있습니다. 여러연구에서 mirna가 protein coding 유전자 30% 이상의발현을조절하는것으로밝혀지고있습니다. mirna의 biogenesis, gene expression regulation mechanism과기능에대한연구가축적되면서복잡한유전자조절네트워크를이해할수있는새로운지평을열었다고인정되고있습니다. 최근연구를통해 mirna는암발생단계나다양한암유형에따라고유한발현양상을보이고있으며많은질병및바이러스감염에중요한역할을할것으로알려지고있습니다. 이러한결과는 mirna가질병진단을위한새로운 biomarker로서기능할뿐아니라 mirna를이용한유전자치료에대한새로운전략을수행할수있다는것을보여줍니다. Figure 1. MicroRNA biogenesis (Annu. Rev. Cell Dev. Biol : ) BIONEER 18

19 AccuTarget mirna mimics & inhibitors Description mirna의안정성을극대화하기위해바이오니아의모든 mirna는 clean room에서제조되며, MALDI-TOF mass spectrometer를통한엄격한 QC 과정을거치고있습니다. 이렇게제조된고효율, 고품질의 mirna는고객의편의를위해주문다음날바로발송해드립니다. 바이오니아의 AccuTarget TM mirna mimic은화학적으로합성되는 double-stranded RNA oligonucleotide로서 mirbase Sequence Database (version 21) 에존재하는 1,700 여개의 human mature mirna와동일한 sequence로제조됩니다. 또한 AccuTarget TM mirna inhibitor는각각의 human mirna를 targeting하여그기능을억제하도록제작되는 single-stranded synthetic RNA입니다. 바이오니아는고객들의요구를충족시키기위해 96-well plate에분주된다양한농도 (0.25, 0.5, 1, 2 nmole) 의 predesigned mimic 및 inhibitor로구성된 mirna mimic, inhibitor library set를제공합니다. 이러한 mirna mimic, inhibitor는 5, 10 및 20 nmole guaranteed yield로주문가능합니다. 또한, 고객이지정한염기서열을가지는 mimic 및 inhibitor도제조가능하며이들로이루어진 flexible mirna library set는최소 48개이상의 mirna로구매가능합니다. Ready-to-transfect mirna mimic은 transfection 후세포에실제존재하는 endogenous mirna와같은활성을나타내며, mirna inhibitor는 target mirna의활성을억제하여 mirna의 loss-offunction 연구에사용될수있습니다. Features and Benefits High Purity, High Quality: Clean room 에서전자동 high-throughput 시스템에의해생산되고, 및 Bio-RP purification 을거친고순도, 고품질의 mirna Affordable pricing: 고품질의다양한 mirna 제품을경제적가격으로제공 Strict QC: MALDI-TOF Mass spectrometry 질량분석데이터가모든 mirna mimic 및 inhibitor 와함께제공되며, PAGE 분석을통해 mirna mimic 의 RNA 두가닥이제대로 annealing 되었는지확인됨 Figure 2. 바이오니아의 AccuTarget TM mirna mimics과타사제품의 reporter gene expression 제어비교평가. HEK cells을 96 well-plate에 100 μl culture media로배양후, transfection 실시. 50~70% confluence한상태에서 10 ng의 luciferase vectors (3 UTR reporter 와 control luciferase) 와 final concentration 100 nm의 mirna mimic을 cotransfection 함. 24시간후, luciferase 활성을측정하여 translational repression 을비교함. mtor와 Lin28b는각각 mir-100과 mir-125b의 validated target mrna. 19

20 AccuTarget mirna mimics & inhibitors Ordering Information AccuTarget TM Custom mirnas Cat. no. Description Purification Guaranteed Yield SMM-001 AccuTarget TM mirna mimic Bio-RP 5 nmole SMM-002 AccuTarget TM mirna mimic Bio-RP 10 nmole SMM-003 AccuTarget TM mirna mimic Bio-RP 20 nmole SMI-001 AccuTarget TM mirna inhibitor Bio-RP 5 nmole SMI-002 AccuTarget TM mirna inhibitor Bio-RP 10 nmole SMI-003 AccuTarget TM mirna inhibitor Bio-RP 20 nmole AccuTarget TM Library mirnas Cat. no. Description Purification Guaranteed Yield SML-1001 AccuTarget TM mirna mimic Bio-RP 0.25 nmole SML-1002 AccuTarget TM mirna mimic Bio-RP 0.5 nmole SML-1003 AccuTarget TM mirna mimic Bio-RP 1 nmole SML-1004 AccuTarget TM mirna mimic Bio-RP 2 nmole SML-2001 AccuTarget TM mirna inhibitor Bio-RP 0.25 nmole SML-2002 AccuTarget TM mirna inhibitor Bio-RP 0.5 nmole SML-2003 AccuTarget TM mirna inhibitor Bio-RP 1 nmole SML-2004 AccuTarget TM mirna inhibitor Bio-RP 2 nmole 단, Flexible library 주문의경우는 minimum 48 EA 이상주문시가능합니다 BIONEER 20

21 AccuTarget mirna Controls Description mirna positive 및 negative control 은바이오니아의 AccuTarget TM mirna 를사용한 mirna 기능연구실험에서대조군으로사용가능합니다. AccuTarget TM mirna Housekeeping Positive Control 은 housekeeping gene 인 GAPDH 의 3 UTR (untranslated region) 을 target 으로하며, transfection control 로사용할수있습니다. mirna negative control sequence 는일반적인 mirna 의구조를가지며 human, mouse, rat 세포주를이용한실험에서 negative control 로사용될수있도록각 genomic sequence 및현재 mirbase database 에있는 mirna sequence 에대해서서열유사성이최소화되어있음을 BLAST 를통해확인하였습니다. 바이오니아는두종류의 mirna negative control 을제공합니다. Features and Benefits Excellent performance: GAPDH를 targeting하여 90% 이상 knockdown efficiency 를나타내는 mirna Housekeeping Positive Control 및 human, mouse, rat에존재하는모든 mirna에대해 minimum sequence identity를갖는 mirna Negative Control High Purity, High Quality: Clean room에서전자동 high-throughput 시스템에의해생산되고, 및 Bio-RP purification을거친고순도, 고품질의 mirna Affordable pricing: 고품질의다양한 mirna 제품을경제적가격으로제공 Figure 3. Performance of mirna mimics positive and negative controls. AccuTarget TM mirna mimic Positive & Negative Controls were transfected at 20 nm using Lipofectamine TM RNAiMAX into HeLa cell lines and assessed for their ability to decrease target mrna levels. Down-regulation of GAPDH was determined using the Real-Time quantitative RT-PCR at 48 hr posttransfection using Bioneer s Exicycler TM 96 qpcr instrument. 21

22 AccuTarget mirna Controls Ordering Information Cat. no. Description Purification Guaranteed Yield SMC-1001 AccuTarget TM mirna Housekeeping Positive Control (GAPDH) Bio-RP 5 nmole SMC-1002 AccuTarget TM mirna Housekeeping Positive Control (GAPDH) Bio-RP 10 nmole SMC-1003 AccuTarget TM mirna Housekeeping Positive Control (GAPDH) Bio-RP 20 nmole SMC-2001 AccuTarget TM mirna mimic Negative Control #1 Bio-RP 5 nmole SMC-2002 AccuTarget TM mirna mimic Negative Control #1 Bio-RP 10 nmole SMC-2003 AccuTarget TM mirna mimic Negative Control #1 Bio-RP 20 nmole SMC-3001 AccuTarget TM mirna mimic Negative Control #2 Bio-RP 5 nmole SMC-3002 AccuTarget TM mirna mimic Negative Control #2 Bio-RP 10 nmole SMC-3003 AccuTarget TM mirna mimic Negative Control #2 Bio-RP 20 nmole SMC-4001 SMC-4002 SMC-4003 SMC-5001 SMC-5002 SMC-5003 AccuTarget TM Fluorescein-labeled mirna mimic Negative Control sirna #1 AccuTarget TM Fluorescein-labeled mirna mimic Negative Control sirna #1 AccuTarget TM Fluorescein-labeled mirna mimic Negative Control sirna #1 AccuTarget TM Fluorescein-labeled mirna mimic Negative Control sirna #2 AccuTarget TM Fluorescein-labeled mirna mimic Negative Control sirna #2 AccuTarget TM Fluorescein-labeled mirna mimic Negative Control sirna #2 5 nmole 10 nmole 20 nmole 5 nmole 10 nmole 20 nmole SMC-2101 AccuTarget TM mirna inhibitor Negative Control #1 Bio-RP 5 nmole SMC-2102 AccuTarget TM mirna inhibitor Negative Control #1 Bio-RP 10 nmole SMC-2103 AccuTarget TM mirna inhibitor Negative Control #1 Bio-RP 20 nmole SMC-3101 AccuTarget TM mirna inhibitor Negative Control #2 Bio-RP 5 nmole SMC-3102 AccuTarget TM mirna inhibitor Negative Control #2 Bio-RP 10 nmole SMC-3103 AccuTarget TM mirna inhibitor Negative Control #2 Bio-RP 20 nmole SMC-4101 SMC-4102 SMC-4103 SMC-5101 SMC-5102 SMC-5103 AccuTarget TM Fluorescein-labeled mirna inhibitor Negative Control sirna #1 AccuTarget TM Fluorescein-labeled mirna inhibitor Negative Control sirna #1 AccuTarget TM Fluorescein-labeled mirna inhibitor Negative Control sirna #1 AccuTarget TM Fluorescein-labeled mirna inhibitor Negative Control sirna #2 AccuTarget TM Fluorescein-labeled mirna inhibitor Negative Control sirna #2 AccuTarget TM Fluorescein-labeled mirna inhibitor Negative Control sirna #2 5 nmole 10 nmole 20 nmole 5 nmole 10 nmole 20 nmole BIONEER 22

23 03 In vivo sirna Custom Service: SAMiRNA In vivo sirna Custom Service Using SAMiRNA TM Technology 24 SAMiRNA TM Technical Spec 26

24 In vivo sirna Custom Service Using SAMiRNA Technology BIONEER Overview 바이오니아의 In vivo sirna Custom Service 는동물모델에서 RNAi 기작을활용한신규 target 발굴, 유전자의기능연구및 in vivo 효능확인에필요한 solution 을제공합니다. Description 바이오니아의풍부한전임상연구경험과 20 여년간전세계에최고품질의올리고와 sirna 를공급해온핵산제조및연구개발노하우가결합된 SAMiRNA TM technology 는독창적인구조의나노입자기반유전자전달기술을바탕으로 RNAi 연구전반에걸친실험서비스를제공합니다. SAMiRNA TM 는특허출원된독창적인구조의자가조립 sirna 나노입자로, 외부의 polymer 층에의해내부 sirna 가보호되는형태입니다. SAMiRNA TM 는 EPR (Enhanced Permeability and Retention) 효과에최적화된나노입자크기로, 표면에부착된 targeting 물질에의해종양조직이나목표장기로선택적으로전달됩니다. 바이오니아는세계적인제약회사들과신약개발을위한 SAMiRNA TM 자체개발연구를지속적으로수행해왔습니다. 차세대 RNAi 신약기술인 SAMiRNA TM 는전세계 RNAi 연구개발자들에게축적된기술과정보를제공하고, 합리적인가격으로 in vivo sirna 연구를수행할수있는새로운기회를제공합니다. SAMiRNA TM (Self-Assembled-Micelle-inhibitory-RNA) SAMiRNA TM 는바이오니아가개발한혁신적인신기술로기존의 sirna 치료제기술의문제점들을극복하여 sirna 를생체내질병표적장기세포까지효율적이고안정적으로전달할수있는나노입자형 RNAi 신약물질입니다. SAMiRNA TM 는세계유일의 sirna prodrug 으로, 단일분자구조스스로나노입자를형성하여주사제로사용되었을때혈액내에서안정적인나노입자구조를유지합니다. 또한, 암및질병조직에만선택적으로전달된후, 세포내에서활성화된 sirna 로전환되어암유발 RNA 를분해시키는획기적인 RNAi 물질입니다. 현재임상테스트에사용중인대부분의 sirna 치료제기술은 sirna 제조후, 제조된 sirna 를생체조직내로전달해주는전달체 (delivery vehicle) 인리포좀 (liposome) 과섞어주는 (formulation 혹은 encapsulation) 과정이있습니다. 이때 sirna 가리포좀안으로들어가는효율 (loading efficiency) 이떨어지고, 낮은 loading efficiency 로인해대량생산시이를분리, 정제하는복잡한 QC 과정이진행됩니다. 또한리포좀의구성성분으로사용되는지질 (lipid) 자체의독성이보고되고있습니다. SAMiRNA TM 는전자동합성라인에서단일분자 (single chemical entity) 로만들어지며 formulation 을거치지않고수용액안에서자가조립되어안정적인나노입자의형태로만들어지기때문에, formulation 을기반으로하는리포좀 sirna 신약기술의문제점을해결할수있습니다. 비임상동물테스트를통해항암효과가입증되었으며일반 sirna 에비해혈액내안정성이뛰어나고, 기존에알려진 sirna 전달체와비교하여독성이매우적어신약후보물질로서뛰어난장점을가지고있습니다. * NOTICE TO PURCHASER: LIMITED LICENSE These products are for research purpose only. They are not for diagnostic or therapeutic purpose and are not to be administered to humans. Use of SAMiRNA is covered by U.S. patents Nos. 8,779,114, 8,771,976, and their foreign counterparts or pending patents. The purchase of these products includes a limited, non-transferable immunity from suit under the forgoing patents for using only this amount of product solely for the purchaser s own internal research. No other patent rights to use this product for any other purpose or for commercial purpose, including without limitation reporting the results of purchaser s activities for a fee or other commercial consideration, are conveyed expressly, by implication, or by estoppels. Further information on purchasing licenses may be obtained by contacting Bioneer at licensing@ bioneer.com SAMiRNA TM is a trademark of Bioneer Corporation. Service Details Custom designed sirna synthesis Custom sirna는고객이제공하는 sirna sequence를합성해드리는서비스입니다. Free-design 서비스는바이오니아에서 sirna 를합성시에만제공되는서비스입니다. 원하시는 sirna sequence 에다양한 modification도가능합니다. 30-mer 이하의 sirna에최대 32 개의서로다른 3 overhang 및다양한옵션을선택하여수많은형태의 sirna/samirna TM 를이용하실수있습니다. Genome-wide predesigned mouse, rat and human sirna 바이오니아의 sirna는고효율이검증된 Turbo si-designer를이용하여설계됩니다. 또한 sirna의안정성을극대화하기위해모든 sirna는 clean room에서제조됩니다. Human, mouse 및 rat에서 44,000개이상의 target 유전자에대한 132,000개의 predesigned sirna가항상준비되기때문에 Search and Order 페이지를이용하여빠르고쉽게 sirna를주문하실수있습니다. 또한 sirna knockdown 실험에꼭필요한검증된 Real-Time PCR primer가모든유전자별로준비되어있습니다. 연구자분들의 RNAi 실험에필요한모든제품들을바이오니아에서 one-stop shopping으로주문할수있습니다. SAMiRNA TM nanoparticle synthesis for in vivo sirna delivery and efficacy/biodistribution tests using animal models 바이오니아에서제공하는 SAMiRNA TM 를이용하여다양한고형암동물모델에주사제로투여하여 target 유전자에대한최적화된효능연구를수행할수있습니다. In vitro screen/validation of sirnas of choice lead optimization service 최적의 target 유전자저해효과를검증하기위해 in vitro sirna/ SAMiRNA screening 및 validation 서비스를제공해드립니다. 세포주대상 100개의 sirna에대하여, 각각 MIQE 가이드라인에준한저해효능검증을수행하여신속하게결과를제공해드리며, 최적의 sirna 효능을갖는 SAMiRNA TM 로합성하여 in vitro에서재확인한후배송해드립니다. 24

25 In vivo sirna Custom Service Using SAMiRNA Technology Quantitation of functional knockdown of target gene by Real-Time qrt-pcr (AccuPower qpcr Array Service) 바이오니아는 20 년간의분자진단연구경험을바탕으로새로운 qpcr Array System 을출시하였습니다. qpcr Array System 은유전자발현분석에있어가장민감하고신뢰성있는방법인 Real- Time PCR 과여러유전자의발현양상을한번에 profiling 할수있는 microarray 분석기법을조합한것으로, 적은비용으로정확한결과를얻을수있는획기적인분석방법입니다. qpcr Array System 은다양한 pathway 와질병에관련된유전자들을한번에 screening 가능하도록 panel 형태로제작되어여러유전자들의발현양상을한눈에볼수있습니다. 또한 qpcr 실험수행시관련된모든과정은논문게재를위한 qpcr 실험지침서인 MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) 가이드라인을따르고있으며, 숙련된전문가들에의해이루어지기때문에정확하고신뢰성있는결과를신속하게얻을수있습니다. Ordering Information 바이오니아의 SAMiRNA TM Custom Service 는다양한서비스패키지를매우경제적인가격으로제공합니다. 수행하고자하는프로젝트의세부사항을 로보내주시면정확한견적및예상소요기간을알려드립니다. * SAMiRNA TM 는주사액에다시녹여즉시동물에투여가능한동결건조형태로제공해드립니다. 입자크기및물질정보에대한 QC 데이터를함께제공해드립니다. SAMiRNA TM Order scale of SAMiRNA TM (nmole) Number of mouse (intravenous injections) 5 mg / kg -> 5 mouse 1 mg / kg -> 25 mouse 5 mg / kg -> 25 mouse 1 mg / kg -> 125 mouse 5 mg / kg -> 50 mouse 1 mg / kg -> 250 mouse 5 mg / kg -> 5 mouse 1 mg / kg -> 25 mouse 5 mg / kg -> 25 mouse 1 mg / kg -> 125 mouse 5 mg / kg -> 50 mouse 1 mg / kg -> 250 mouse 5 mg / kg -> 5 mouse 1 mg / kg -> 25 mouse Price ( 원 ) 100 Custom Designed SAMiRNA TM 500 Inquire Control SAMiRNA TM 500 Inquire 1000 Fluorescence conjugated SAMiRNA TM (FITC, Cy5.5 etc) 100 Inquire 25

26 SAMiRNA Technical Spec Description SAMiRNA TM 는효율은극대화하고부작용은최소화한차세대 RNAi 플랫폼기술로서, 이를이용하여기존에약이존재하지않았던난치성질병에대한 sirna 치료제개발이가능합니다. Pre-clinical Data for SAMiRNATM 1. 동물모델에서의 SAMiRNA TM 항암효과 RNAi 기반신약개발의모든핵심과제들을해결 기존 sirna 기술의주요문제점생체내불안정독성유발 표적세포내전달효율낮음 낮은유전자저해효율 SAMiRNA TM 전구약물기술탁월한생체내안정성독성이나부작용없음생체내질병표적장기 / 암세포로효율적인전달동물모델에서낮은투여량으로도지속적약효 Figure 2. SAMiRNA TM 나노입자의타켓 mrna in vivo 발현저해효과. (A) 종양이이식된 mouse 모델에미정맥으로 PBS와 SAMiRNA, 종양표적물질인엽산 (FA, folic acid) 이결합된 SAMiRNA TM 나노입자를 5 mg/kg으로단회투여한뒤, 표시된시간이경과하였을때종양조직을채취하여조직안의 target 유전자인 survivin의 mrna 양을 Real-Time PCR로측정함. (B) 종양이이식된 mouse 모델에미정맥으로 PBS와 SAMiRNA TM 를 1 mg/kg 또는 5 mg/kg으로단회투여한뒤, 70 시간이경과하였을때종양조직을채취하여조직안의 target 유전자인 survivin의 mrna의양을 Real-Time PCR로측정함. 2. 암세포특이적전달특성 신개념고효율 RNAi 신약기술 Figure 1. SAMiRNA TM 나노입자의물리화학적특성. (A) SAMiRNA TM 의도식화. (B) SAMiRNA TM 의동결전자현미경 (Cryo-TEM) 사진 (scare bar = 100 nm). Figure 3. In vivo targeting of SAMiRNA TM Nanoparticles to s.c. grafted tumor. (A) 형광 (Cy5.5) 표지된 SAMiRNA TM 나노입자의 in vivo 종양전달특이성확인. 종양이이식된마우스모델에미정맥으로투여함. (B) 48시간경과시점에서채취된장기에서측정된 SAMiRNA TM 의형광이미지. 종양조직을제외한타장기에서는형광이측정되지않음. BIONEER 26

27 SAMiRNA Technical Specs 3. 탁월한생체내안정성 SAMiRNA TM 투여후, 혈청안정성및약동력학 (PK/PD) 확인. *Human blood cell Figure 4. SAMiRNA TM i.v injection 후 mouse에서혈청을채취하여 HIT qrt- PCR 방법을이용하여 sirna 검출. sirna PK/PD 확인을위하여 SAM-siRNA를 ICR mouse에미정맥투여후, 0.5, 2, 4, 6, 10, 24, 48, 72 시간에혈액을추출하여 plasma에서 SAMiRNA TM 와siRNA detection 진행. 투여량과, 투여후경과시간의존적으로 SAMiRNA TM 와 sirna양이감소하고, SAMiRNA TM 의경우 72 시간까지 detection됨을확인. Figure 6. Human 혈액세포에서 SAMiRNA TM 투여에의한 innate immune response 유발확인. Human blood sample을채혈하여 PBMC세포를분획한뒤 culture dish에분주함. 각군의 SAMiRNA TM sample을각 well 에분주하고나서 37 에서 24시간반응시킨뒤상층액을모아서공인인증기관에서 cytokine induction 유무를 ELISA 를통해측정. 4. 부작용이없는뛰어난안전성 *Mouse Figure 5. SAMiRNA TM 투여에따른간독성, 일반독성, cytokine 측정결과. 생후 5주된 ICR 생쥐에 None (not-injected), PBS, TF (QBiogene 사의 in vivo MegaFectine), β- gal 728 with TF, NC sirna (negative control sirna), Sur584 (survivin sirna), SAM-siCON (negative control sirna-containing SAMiRNA TM ), SAM-sur584 (survivin sirna-containing SAMiRNA TM ) 의각물질을 5 mg/kg의농도로투여한뒤 6시간후에비장을채취하여균질화한후, mrna 를추출하여 cytokine mrna level을 qpcr분석함. 27

28 SAMiRNA Technical Specs *28일간반복투여후, SAMiRNA-Survivin의독성확인 1) 일반독성시험 (a) 단회정맥투여독성시험 - SAMiRNA TM 를암수마우스 (ICR) 에단회정맥투여후발현되는급성독성을조사하기위해실시. 군당암수각각 5마리씩을이용하여시험물질을 O ( 부형제대조군 ) 및 100 mg/kg의용량으로 1 회정맥투여한후 15 일간사망률, 일반증상, 체중측정및부검소견관찰을실시. (1) 시험기간중사망동물은발생하지않았음. (2) 일반증상관찰결과, 시험물질투여와관련된증상은관찰되지않았음. (3) 체중측정결과, 시험물질투여와관련된변화는관찰되지않았음. (4) 부검결과, 시험물질투여와관련된이상소견은관찰되지않았음. (b) 4주반복정맥투여독성시험 - SAMiRNA TM 를암수마우스 (ICR) 에 4주간매일 1회반복하여정맥으로투여하였을때발현되는독성을조사하기위해실시. 군당암수각각 10 마리씩을이용하여시험물질을 0 ( 부형제대조군 ), 25, 50 및 100 mg/kg의용량으로 4주간투여한후사망률및일반증상을관찰하였고, 체중측정, 사료섭취량측정, 안검사, 혈액학적검사, 혈액생화학적검사, 뇨검사, 부검소견관찰, 장기중량측정및조직병리학적검사를실시. (1) 시험기간중시험물질투여와관련된사망동물은발생하지않았음. (2) 일반증상관찰결과, 암수모든투여군에서시험물질투여와관련된증상은관찰되지않았음. (3) 체중측정결과, 암수모든투여군에서시험물질투여와관련된변화는관찰되지않았음. (4) 사료섭취량측정결과, 암수모든투여군에서시험물질투여와관련된변화는관찰되지않았음. (5) 안검사결과, 암수모든투여군에서시험물질투여와관련된변화는관찰되지않았음. (6) 혈액학적검사결과, 암컷 100 mg/kg/day 투여군에서혈소판수치가감소하였음. (7) 혈액생화학적검사결과, 암수모든투여군에서시험물질투여와관련된변화는관찰되지않았음. (8) 뇨검사결과, 암수모든투여군에서시험물질투여와관련된변화는관찰되지않았음. (9) 조직병리학적검사결과, 시험물질투여와관련된이상소견은관찰되지않았음. - SAMiRNA TM 를 4주간정맥투여하였을때무해용량 (NOAEL: No Observed Adverse Effect Level) 은암수모두 100 mg/ kg/day로확인. 5. 안전성평가 SAMiRNA TM 는일반독성시험 ( 단회, 4 주반복정맥투여독성시험 ) 및유전독성시험 ( 복귀돌연변이, 체외염색체이상시험 ) 을통해서신약후보물질로써우수한안전성이입증됨. Figure 7. 공인인증기관분석을통한 SAMiRNA TM 독성 Profile. SAMiRNA TM 를생후 6주된마우스 (ICR) 에 3일간매일 1회반복하여정맥으로투여하였을때발현되는독성을조사하기위해실시. SAMiRNA TM 은 10 mg/kg의농도로투여하였으며마지막투여 24시간후에채혈을통해전혈 (whole blood) 및혈청 (serum) 을얻어혈액생화학적검사 (Blood chemical analysis) 및혈액학적검사 (Hematological analysis) 를수행하였음. (1) 혈액생화학적검사결과, 시험물질투여와관련된변화는관찰되지않았음. (2) 혈액학적검사결과, 시험물질투여와관련된변화는관찰되지않았음. BIONEER 2) 유전독성시험 (a) 복귀돌연변이시험 (Ames test) - SAMiRNA TM 은 5,000 μg/plate 농도에이르기까지 Salmonella typhimurium 4종및 Escherichia coli 1종시험균주에복귀돌연변이를유발하지않았음. (b) 체외염색체이상시험 (Chromosome aberration assay) - SAMiRNA TM 은대사활성계적용여부에관계없이 CHL 세포 (Chinese Hamster Lung cell) 에염색체이상을유발하지않았음. 28