siPOOLs用于藥物靶點(diǎn)篩選研究（RNAi）

對于靶向基因沉默，RNA干擾（RNAi)具有易于操作、快速結(jié)果、高效率、廣泛適用于各種細(xì)胞類型的多重優(yōu) 勢。其具有瞬轉(zhuǎn)效應(yīng)和劑量依賴效應(yīng)，這點(diǎn)與小分子非常相似。然而，目前的siRNA試劑其特異性和基因沉默 效率可變，阻礙了它們作為藥物發(fā)現(xiàn)和基因研究工具的應(yīng)用。

siTOOLs Biotech由Michael Hannus博士和Gunter Meister教授于2013年9月成立，由德國雷根斯堡大學(xué) 和Intana Bioscience制藥服務(wù)公司聯(lián)合運(yùn)營。目前推出有3個(gè)產(chǎn)品系列：（1）siPOOLs：一種siRNA混合物，有 效增加基因沉默特異性，“稀釋”傳統(tǒng)RNA干擾試劑的脫靶效應(yīng)。（2）raPOOLs：一種穩(wěn)健的RNA親和純化方 案，適用于基因功能和相互作用的生物化學(xué)研究。（3）riboPOOLs：適合任意物種的核糖體RNA去除試劑，高效且經(jīng)濟(jì)。

可靠表型的特異性基因沉默工具

siPOOLs是一款經(jīng)過優(yōu)化設(shè)計(jì)的高復(fù)雜性的包含30條siRNA的混合物，經(jīng)證明可有效消除脫靶效應(yīng)，并提高結(jié)果的可靠性（Hannus et al., 2014）。 Pack Hunter (pooling) 方法通過將當(dāng)個(gè)siRNA的 濃度稀釋到刺激表型的閾值以下來對抗單個(gè)siRNA的脫靶。 借助專有的設(shè)計(jì)算法，siPOOLs中的siRNA序列經(jīng) 過優(yōu)化，以實(shí)現(xiàn)最大的轉(zhuǎn)錄本覆蓋率，高效雜交并 對旁系同源基因進(jìn)行過濾，從而實(shí)現(xiàn)高效和特異性 的基因沉默。

siPOOLs產(chǎn)品優(yōu)勢

使用簡單快捷：siPOOLs與多種轉(zhuǎn)染試劑兼容，幾天內(nèi)就能看 到結(jié)果。

高度特異性且有效：siPOOLs在標(biāo)準(zhǔn)細(xì)胞系中將脫靶率降低5-25 倍，且在1-3 nM下實(shí)現(xiàn)基因敲低率≥70%。

一致的表型：與siRNA相反，由序列非依賴性的siPOOL產(chǎn)生 的表型高度一致。

確保經(jīng)過檢驗(yàn)：通過RT-qPCR進(jìn)行siPOOL驗(yàn)證，如果在 轉(zhuǎn)染下敲低率低于70%，則有可能重新設(shè)計(jì)。

使用注釋進(jìn)行定制設(shè)計(jì)：專業(yè)的設(shè)計(jì)，確保優(yōu)化熱力學(xué)特性且避免旁系 同源基因。

HPLC純化且無毒：所有siPOOL均經(jīng)過HPLC純化，可降低污染物 和副作用的風(fēng)險(xiǎn)。

關(guān)鍵問題：siRNAs的脫靶效應(yīng)

科學(xué)家們一直在使用RNA干擾（RNAi）作為研究基因功能的快速有效的工具。然而，短干擾RNA（siRNA）的脫靶效應(yīng)和可變性能仍然是一個(gè)令人頭疼的缺點(diǎn)，在驗(yàn)證工作中消耗了寶貴的時(shí)間和資源。

siRNA通常與靶RNA轉(zhuǎn)錄本互補(bǔ)結(jié)合，通過RNAi機(jī)制指導(dǎo)其降解。脫靶效應(yīng)主要是由siRNA模擬內(nèi)源性 基因調(diào)節(jié)因子microRNA（miRNA）引起的。由于miRNA只需要6個(gè)堿基種子匹配到3'非翻譯區(qū)（UTR）即可觸 發(fā)轉(zhuǎn)錄本下調(diào)，因此siRNA在通過這種機(jī)制處理時(shí)可以改變許多意外靶標(biāo)的表達(dá)。

siPOOLs如何提高特異性

單個(gè)siRNA或含有3-4個(gè)siRNA的低復(fù)雜度siRNA庫，經(jīng)常擊中多個(gè)脫靶基因并表現(xiàn)出易變的靶基因敲低。 siPOOL是高度復(fù)雜且確定的30個(gè)siRNA池，每個(gè)siRNA以皮摩爾工作濃度存在。因此， · 稀釋了每個(gè)siRNA的脫靶特征，提高了靶向特異性。 · 確保了靶基因的協(xié)同敲低，產(chǎn)生更穩(wěn)健、更可靠的結(jié)果。

使用siPOOL具有更高的特異性

特異性好的試劑應(yīng)僅影響其靶標(biāo)基因。

HeLa細(xì)胞中微陣列的表達(dá)譜分析顯示，單個(gè)siRNA可以誘導(dǎo)許多脫靶基因（紅點(diǎn)），而針對同一靶基因（綠 點(diǎn)）并包含非特異性siRNA的siPOOL大大降低了脫靶效應(yīng)。

通過siPOOL實(shí)現(xiàn)更好的重現(xiàn)性和有效的敲低效率

普通siRNA的敲低效率差異很大。

與單個(gè)siRNA相比，針對同一基因的siPOOLs具有相似的敲低效率，表明具有更高的穩(wěn)健性和可重復(fù)性（左 圖和中圖：靶RNA水平的實(shí)時(shí)定量PCR測量的相關(guān)圖）。 siPOOLs也表現(xiàn)出有效的基因敲低。在常用的細(xì)胞系中，許多基因通常在1 nM濃度下實(shí)現(xiàn)75-98%的基 因敲低效率（右圖）。

您可以信賴的表型

如果RNAi效果可靠且特異，靶向同一基因的兩種試劑應(yīng)產(chǎn)生相似的表型。

每個(gè)基因分別使用兩個(gè)siPOOL池，一個(gè)來自我們的人類激酶siPOOL文庫，一個(gè)是市售的每個(gè)基因含3條 siRNAs的文庫，科學(xué)家在A549細(xì)胞中篩選了36個(gè)基因并檢測了細(xì)胞活力。僅siPOOLs產(chǎn)生了較好的一致性表型。

siTOOLs生物技術(shù)始終致力于幫助學(xué)術(shù)研究人員、科學(xué)家、制藥/生物技術(shù)公司和RNAi篩選人員。目前，已幫助許多論文 發(fā)表在《自然》、《細(xì)胞》、《自然醫(yī)學(xué)》、《科學(xué)報(bào)告》等頂級期刊上。期待與您的合作，我們將為您的RNA研究提供量身定制 的分子工具。

參考文獻(xiàn)

Hannus M. et al. (2014) siPools: highly complex but accurately defined siRNA pools eliminate off-target effects. Nucleic Acids Res 42(12): 8049-61

Zimmermann et al. (2023) Cold Atmospheric Plasma Triggers Apoptosis via the Unfolded Protein Response in Melanoma Cells MDPI

Müller et al. (2023) Plakophilin 3 facilitates G1/S phase transition and enhances proliferation by capturing RB protein in the cytoplasm and promoting EGFR signaling Cell Reports

Luo et al. (2023) The long non-coding RNA LINC00958 is induced in psoriasis epidermis and modulates epidermal proliferation Journal of Investigative Dermatology

Staebler et al. (2023) MIA/CD-RAP Regulates MMP13 and Is a Potential New Disease-Modifying Target for Osteoarthritis Therapy MDPI

Hertel et al. (2022) USP32-regulated LAMTOR1 ubiquitination impacts mTORC1 activation and autophagy induction Cell Reports

Yavon et al. (2022) High-throughput morphometric and transcriptomic profiling uncovers composition of naïve and sensory-deprived cortical cholinergic VIP/CHAT neurons The EMBO Journal

Kim et al. (2022) Screening of the siGPCR library in combination with cisplatin against lung cancers Scientific Reports

Mitani et al. (2022) SNAP23-Mediated Perturbation of Cholesterol-Enriched Membrane Microdomain Promotes Extracellular Vesicle Production in Src-Activated Cancer Cells Biological and Pharmaceutical Bulletin

Blümel et al. (2022) Primary cilia contribute to the aggressiveness of atypical teratoid/rhabdoid tumors Cell Death & Disease

Rovira et al. (2022) The lysosomal proteome of senescent cells contributes to the senescence secretome Aging Cell

Koch et al. (2022) Targeting the Retinoblastoma/E2F repressive complex by CDK4/6 inhibitors amplifies oncolytic potency of an oncolytic adenovirus Nature Communications

Schaefer et al. (2022) Global and precise identification of functionalmiRNA targets in mESCs by integrative analysis EMBO Reports

Kerstin Dörner et al. (2022) Genome-wide RNAi screen identifies novel players in human 60S subunit biogenesis including key enzymes of polyamine metabolism Nucleic Acids Research

Bartl et al. (2022) The HHIP-AS1 lncRNA promotes tumorigenicity through stabilization of dynein complex 1 in human SHH-driven tumors Nature Communications

Banas et al. (2022) The Interplay of NEAT1 and miR-339-5p Influences on Mesangial Gene Expression and Function in Various Diabetic-Associated Injury Models non-coding RNA

Mestre-Farràs et al. (2022) Melanoma RBPome identification reveals PDIA6 as an unconventional RNA-binding protein involved in metastasis Nucleic Acids Research

Dessauges et al. (2022) Optogenetic actuator – ERK biosensor circuits identify MAPK network nodes that shape ERK dynamics Molecular Systems Biology

Falke et al. (2022) Knockdown of the stem cell marker Musashi-1 inhibits endometrial cancer growth and sensitizes cells to radiation Stem Cell Research & Therapy

Gutierrez-Prat et al. (2022) DUSP4 protects BRAF- and NRAS-mutant melanoma from oncogene overdose through modulation of MITF Life Science Alliance

Lechner et al. (2022) Target deconvolution of HDAC pharmacopoeia reveals MBLAC2 as common off-target Nature Chemical Biology

Daniel Kummer et al. (2022)  A JAM-A–tetraspanin–αvβ5 integrin complex regulates contact inhibition of locomotion Journal of Cell Biology

Delicato, A. et al (2022) YB-1 Oncoprotein Controls PI3K/Akt Pathway by Reducing Pten Protein Level Genes

Szachnowski et al. (2022) Transcriptomic landscapes of SARS-CoV-2-infected and bystander lung cells reveal a selective upregulation of NF-κB-dependent coding and non-coding proviral transcripts bioRxiv - preprint

Hauffe, L. et al. (2022) Eukaryotic translation initiation factor 4E binding protein 1 (EIF4EBP1) expression in glioblastoma is driven by ETS1- and MYBL2-dependent transcriptional activation Cell Death Discovery

Till Braun et al.  (2022) Non-canonical function of AGO2 augments T-cell receptor signaling in T-cell prolymphocytic leukemia Amercian Association for Cancer Research

Karabid et al.  (2022) Angpt2/Tie2 autostimulatory loop controls tumorigenesis EMBO Mol Med

Kerstin Dörner et al. (2022) Genome-wide RNAi screen identifies novel players in human 60S subunit biogenesis including key enzymes of polyamine metabolism Nucleic Acids Research

X. Li et al.  (2022) 5’isomiR-183-5p|+2 Elicits Tumor Suppressor Activity in a Negative Feedback Loop with E2F1 Journal of Experimental & Clinical Cancer Research - preprint -

Thölmann et al. (2022) JAM-A interacts with α3β1 integrin and tetraspanins CD151 and CD9 to regulate collective cell migration of polarized epithelial cells Cellular and Molecular Life Sciences

Thea Reinkens et al. (2021) Ago-RIP Sequencing Identifies New MicroRNA-449a-5p  Target Genes Increasing Sorafenib Efficacy in  Hepatocellular Carcinoma Journal Of Cancer

Weihua Qin et. Al (2022) Probing protein ubiquitination in live cells bioRxiv - preprint

Belitškin et al. (2021) Hepsin regulates TGFβ signaling via fibronectin proteolysis EMBO reports

Q. Zou et al (2021) Utility of single-shot compressed sensing cardiac magnetic resonance cine imaging for assessment of biventricular function in free-breathing and arrhythmic pediatric patients International Journal of Cardiology

J. Laisney et al (2021) Delivery of short hairpin RNA in the neotropical brown stink bug, Euschistus heros, using a composite nanomaterial Pesticide Biochemistry and Physiology

Indacochea et al (2021) Cold-inducible RNA bindi ng protein promotes breast cancer cell maligna ncy by regulating Cys tatin C levels Cold Spring Harbor Laboratory Press

Khan et al (2021) Developing Tumor Radiosensitivity Signatures Using LncRNAs Radiation Research

Ghodke et al (2021) AHNAK controls 53BP1-mediated p53 response by restraining 53BP1 oligomerization and phase separation Molecular Cell

Gaza et al (2021) Identification of novel targets of miR-622 in hepatocellular carcinoma reveals common regulation of cooperating genes and outlines the oncogenic role of zinc finger CCHC-type containing 11 Neoplasia

Indacochea et al (2021) Cold-inducible RNA binding protein promotes breast cancer cell maligna ncy by regulating Cys tatin C levels Cold Spring Harbor Laboratory Pres

Mercier et al (2020) Endosomal membrane tension regulates ESCRT-III-dependent intra-lumenal vesicle formation Nature Cell Biology

Ghosh et al (2020) Prevention of dsRNA-induced interferon signaling by AGO1x is linked to breast cancer cell proliferation The EMBO Journal

Dhamija et. al (2020) A pan-cancer analysis reveals nonstop extension mutations causing SMAD4 tumour suppressor degradation Nature Cell Biology

Gandhi et. al (2020) The lncRNA lincNMR regulates nucleotide metabolism via a YBX1 - RRM2 axis in cancer Nature Communications

Stephan et. al (2020) MICOS assembly controls mitochondrial inner membrane remodeling and crista junction redistribution to mediate cristae formation The EMBO journal

Liebig et. al (2020) HuRdling Senescence: HuR Breaks BRAF-Induced Senescence in Melanocytes and Supports Melanoma Growth. MDPI Cancers

Prasad et. al (2020) The UPR sensor IRE1α and the adenovirus E3-19K glycoprotein sustain persistent and lytic infections. Nature Communications

Marchetto et. al (2020)  Oncogenic hijacking of a developmental transcription factor evokes vulnerability toward oxidative stress in Ewing sarcoma. Nature Communications

Larios, J. et. al (2020) ALIX- and ESCRT-III–dependent sorting of tetraspanins to exosomes. JCB

Mahli, A. et. al (2019) Bone Morphogenic Protein-8B Expression is Induced in Steatotic Hepatocytes and Promotes Hepatic Steatosis and Inflammation in Vitro. Cells

Renner, K. et. al (2019) Restricting Glycolysis Preserves T Cell Effector Functions and Augments Checkpoint Therapy. Cell Rep

Werner, S. et. al (2019) MRTF-A controls myofibroblastic differentiation of human multipotent stromal cells and their tumour-supporting function in xenograft models. Scientific Reports

Braun, C. et. al (2019) Inhibition of peptidyl-prolyl isomerase (PIN1) and BRAF signaling to target melanoma. Am J Transl Res

Kappelmann-Fenzl, M. et. al (2019) C-Jun drives melanoma progression in PTEN wild type melanoma cells. Cell Death Dis

Simchovitz, A et. al (2019) NEAT1 is overexpressed in Parkinson’s disease substantia nigra and confers drug-inducible neuroprotection from oxidative stress. FASEB J

Mahli, A et. al (2019) Bone Morphogenetic Protein-8B Expression is Induced in Steatotic Hepatocytes and Promotes Hepatic Steatosis and Inflammation In Vitro. Cells

Dietrich, P et. al (2019) The Delta Subunit of Rod-Specific Photoreceptor cGMP Phosphodiesterase (PDE6D) Contributes to Hepatocellular Carcinoma Progression. Cancers

Feuerer, L et. al (2019) Role of MIA (melanoma inhibitory activity) in melanocyte senescence. Pigment Cell Melanoma Res

Ziegler, C. et. al (2019) The long non‐coding RNA LINC00941 and SPRR5 are novel regulators of human epidermal homeostasis. EMBO Rep

Choi, K. et. al (2019) Binary Targeting of siRNA to Hematologic Cancer Cells In Vivo Using Layer‐by‐Layer Nanoparticles. Adv Funct Mater

Spies, J. et. al (2019) 53BP1 nuclear bodies enforce replication timing at under-replicated DNA to limit heritable DNA damage. Nat Cell Biol

Dietrich, P. et al. (2019) Neuroblastoma RAS Viral Oncogene Homolog (NRAS) Is a Novel Prognostic Marker and Contributes to Sorafenib Resistance in Hepatocellular Carcinoma. Neoplasia

Vendramin, R. et al. (2018) SAMMSON fosters cancer cell fitness by concertedly enhancing mitochondrial and cytosolic translation. Nat Struct Mol Biol

Coscia, F. et al. (2018) Multi-level Proteomics Identifies CT45 as a Chemosensitivity Mediator and Immunotherapy Target in Ovarian Cancer. Cell

Kästle, M. et al. FKBP51 modulates steroid sensitivity and NFκB signalling: A novel anti-inflammatory drug target. Eur J Immunol

Stieglitz, D. et al. BMP6-induced modulation of the tumor micro-milieu. Oncogene 10.1038/s41388-018-0475-x (2018)

Weber, A., Schwarz, S. C. et al. Epigenome-wide DNA methylation profiling in Progressive Supranuclear Palsy reveals major changes at DLX1. Nat Commun 9, 2929 (2018)

Noethel, B. et al. Transition of responsive mechanosensitive elements from focal adhesions to adherens junctions upon epithelial differentiation. Mol Biol Cell 10.1091/mbc.E17-06-0387(2018)

Prestigiacomo, V., and Suter-Dick, L.  Nrf2 protects stellate cells from Smad-dependent cell activation. PLoS One 13, e0201044 (2018)

Vallin, B. et al. Novel short isoforms of adenylyl cyclase as negative regulators of cAMP production. Biochim Biophys Acta - Mol Cell Res 1865: 1326–1340 (2018)

Rietscher, K. et al.14-3-3 Proteins Regulate Desmosomal Adhesion via Plakophilins.” Journal of cell science 131(10). (2018).

Jiang, P. et al. Genome-Scale Signatures of Gene Interaction from Compound Screens Predict Clinical Efficacy of Targeted Cancer Therapies.” Cell Systems 6(3): 343–354.e5.  (2018).

So, D. et al. Cervical Cancer Is Addicted to SIRT1 Disarming the AIM2 Antiviral Defense. Oncogene (2018).

Klingenberg, M. et al. The lncRNA CASC9 and RNA Binding Protein HNRNPL Form a Complex and Co-Regulate Genes Linked to AKT Signaling. Hepatology (2018).

Polycarpou-Schwarz, M. et al. The Cancer-Associated Microprotein CASIMO1 Controls Cell Proliferation and Interacts with Squalene Epoxidase Modulating Lipid Droplet Formation. Oncogene (2018)

Zeiner, P. S. et al. CD74 regulates complexity of tumor cell HLA class II peptidome in brain metastasis and is a positive prognostic marker for patient survival. Acta Neuropathol. Commun. 6, 18 (2018).

Ziegelmann, B et al. Lithium chloride effectively kills the honey bee parasite Varroa destructor by a systemic mode of action. Scientific Reports 8(1), 683 (2018)

Dietrich, P. et al. Wild type Kirsten rat sarcoma is a novel microRNA-622-regulated therapeutic target for hepatocellular carcinoma and contributes to sorafenib resistance. Gut (2017)

Dietrich, P. et al. Wild-type KRAS is a novel therapeutic target for melanoma contributing to primary and acquired resistance to BRAF inhibition. Oncogene (2017)

Kaller, M. et al. Loss of p53-inducible long non-coding RNA LINC01021 increases chemosensitivity. Oncotarget 8, 102783–102800 (2017).

Ilina, E. I. et al. Effects of soluble CPE on glioma cell migration are associated with mTOR activation and enhanced glucose flux. Oncotarget 8, 67567–67591 (2017).

Goyal, A. et al. A cautionary tale of sense-antisense gene pairs: independent regulation despite inverse correlation of expression. Nucleic Acids Research (2017)

Steinbach, A. et al. ERAP1 overexpression in HPV-induced malignancies: A possible novel immune evasion mechanism. Oncoimmunology 6, e1336594 (2017).

Sluch, V. M. et al. Enhanced Stem Cell Differentiation and Immunopurification of Genome Engineered Human Retinal Ganglion Cells. STEM CELLS Translational Medicine. (2017) doi:10.1002/sctm.17-0059

Armento, A. et al. Carboxypeptidase E transmits its anti-migratory function in glioma cells via transcriptional regulation of cell architecture and motility regulating factors. International Journal of Oncology 51(2), 702-714 (2017)

Nötzold, L. et al. The long non-coding RNA LINC00152 is essential for cell cycle progression through mitosis in HeLa cells. Scientific Reports 7, 2265 (2017)  Also cites raPOOL

Welsbie, D. S. et al. Enhanced Functional Genomic Screening Identifies Novel Mediators of Dual Leucine Zipper Kinase-Dependent Injury Signaling in Neurons. Neuron 94(6), 1142–1154.e6 (2017)

Ruivo, M. T. G. et al. Host AMPK Is a Modulator of Plasmodium Liver Infection. Cell Rep. 1–7 (2017).

Treiber, T. et al. A Compendium of RNA-Binding Proteins that Regulate MicroRNA Biogenesis. Mol. Cell 66, 270–284.e13 (2017).

Seiler, J. et al. The lncRNA VELUCT strongly regulates viability of lung cancer cells despite its extremely low abundance. Nucleic Acids Res. (2017)

Goyal, A. et al. Challenges of CRISPR/Cas9 applications for long non-coding RNA genes. Nucleic Acids Res. (2016)

Ott, C. A. et al. Induction of exportin-5 expression during melanoma development supports the cellular behavior of human malignant melanoma cells. Oncotarget 7, 62292–62304 (2016).

Adriaens, C. et al. p53 induces formation of NEAT1 lncRNA-containing paraspeckles that modulate replication stress response and chemosensitivity. Nat. Med. (2016).

Coll-Bonfill, N. et al. Slug is increased in vascular remodeling and induces a smooth muscle cell proliferative phenotype. PLoS One 11, 1–21 (2016).

Kordaß, T. et al. SOX5 is involved in balanced MITF regulation in human melanoma cells. BMC Med. Genomics 9, 10 (2016).

Hauptmann, J. et al. Biochemical isolation of Argonaute protein complexes by Ago-APP. Proc. Natl. Acad. Sci. 112, 11841–11845 (2015).

Blirando, K. et al. The stellate vascular smooth muscle cell phenotype is induced by IL-1β via the secretion of PGE2 and subsequent cAMP-dependent protein kinase A activation. Biochim. Biophys. Acta. 1853, 3235–3247 (2015).

Tuncay, H. et al. JAM-A regulates cortical dynein localization through Cdc42 to control planar spindle orientation during mitosis. Nat. Commun. 6, 8128 (2015).

Schraivogel, D. et al. Importin-β facilitates nuclear import of human GW proteins and balances cytoplasmic gene silencing protein levels. Nucleic Acids Res. (2015)

Schönemann L. et al.: Reconstitution of CPSF active in polyadenylation: recognition of the polyadenylation signal by WDR33. Genes Dev. 28(21), 2381-93 (2014)

Prasad, V. et al. Chemical induction of unfolded protein response enhances cancer cell killing through lytic virus infection. J. Virol. 88, 13086–13098 (2014).

Patrick C.H. Lo, PhD: siRNAs: Jumping in the Pool to Avoid Hitting Innocent Bystanders. BioTechniques (2014)

Thalyana S. Advances in RNAi Tools and Technologies. Genetic Engineering & Biotechnology News. April 33(8): 20-22, 24 (2013)

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