ARN interférence Technologies : sirna, shrna, mirna

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1 ARN interférence Technologies : sirna, shrna, mirna LabCluster Tour Delphine Ayache sigma-aldrich.com

2 Methods for Modulating Gene Expression DNA RNA Protein Transcription Translation Genome Editing: Zinc Finger Nucleases CRISPR RNA Interference: sirna esirna shrna mirna Small Molecules mabs sigma-aldrich.com 2

3 Agenda RNA interference (RNAi) : sirna shrna Detecting knockdown mirna sigma-aldrich.com 3

4 RNA interference (RNAi) RNAi refers to dsrna-induced gene silencing Role in : defending cells against parasitic genes viruses and transposons directing development as well as gene expression. Uses RNAi to suppress the expression of interesting genes sigma-aldrich.com 4

5 sigma-aldrich.com RNAi tools : sirna

6 RNAi : small interfering RNA (sirna) Small synthetic RNA duplexes : complementary base pairs and 2-nucleotide 3 overhangs Transfected into cells or injected into animals : Typical gene silencing activities peaking around hours and is 5-7 days in duration, depending on transfection efficiency, cellular growth rate and protein turnover. Recommended for easy to transfect cells Simple, transient sigma-aldrich.com 6

7 sirna process sirna is processed by Dicer sirna assembles with RISC and unwinds sirna mediated target recognition mrna cleavage and degradation sigma-aldrich.com 7

8 Pre-designed and custom sirna Pre-designed human, mouse and rat sirna (Rosetta Inpharmatics Algorithm) : Guaranteed 75% knockdown on 2-out-of-3 sequences per gene Individual sirna, allowing for optional pooling steps (3 to 4 sirna) Panels, libraries Custom sirna (your sequence): Publications In-house design Lower cost than pre-designed sirna sirna negative controls Universal negative controls #1 and #2 : No homology to all mature and predicted RefSeq mrna sequences in human, mouse and rat Scrambled sirna : random mix of your best sirna sequence; Check the BLAST! Gilot, D. et al RNAi-Based Screening Identifies Kinases Interfering with Dioxin-Mediated Up- Regulation of CYP1A1 Activity. PLoS ONE 6(3): e18261 Sigma.com/missionsirna sigma-aldrich.com 8

9 Design Specificity: Critical Nucleotide Positions in sirna Guide strand regions 5-3 prime: The very 5 prime end is critical for RISC strand selection and entry into the RISC Position 2-8 is the seed region : has been shown to determine specificity. Position 10: Cleavage occurs between nucleotides 10 and 11 from 5 end of guide strand Position 10-16: cleavage specificity, mismatches within this region produces highly discriminatory sirnas. Fluorophore : 3 guide or 5 non guide sigma-aldrich.com D. Bumcrot et al., (2006) Nat. Chem. Biol. 2, 711

10 Main sirna rules Avoid sirna degradation (ribonucleases, repeated freeze/thaw cycles, dry / liquid) sirna dilution by cell divisions depends of the cell type and physiological conditions Importance of knocked down functions for cell life and cell division Half -life and amount of protein in the cell : the persistence of the proteins may delay the coming out of silencing even with mrna knock down sigma-aldrich.com 10

11 RNAi : Endoribonuclease prepared sirna (esirna) Selection of the target region Generation of corresponding cdna in vitro transcription to generate long dsrna Digestion with Dicer or RNase III into super pools of sirna Purification Super pool of hundred of sirnas against one target gene. Lower off target effects than single or pooled sirna Very low cost Guaranteed 70% knockdown 16,744 human and 14,068 mouse targets Quick primary screens, validation sigma.com/esirna sigma-aldrich.com 11 Kittler et al. (2007) Nat. Methods 4,

12 esirna versus sirna sigma-aldrich.com 12

13 esirnas benefits Decrease of off-target effects Kittler et al, NATURE METHODS VOL.4 NO.4 APRIL sigma-aldrich.com 13

14 esirna : knock down efficiency Sigma Bioguarantee: KD minimum 70% sigma-aldrich.com 14

15 sigma-aldrich.com RNAi tools : shrna

16 RNAi : short hairpin RNA (shrna) shrna are precursors of sirnas single bs strand : two complementary sequence anneal intra-molecularly + a non relevant loop sequence form a hairpin. The endogenous Dicer enzyme recognizes and cleaves the RNA structure into the desired sirna molecule. duplexes : complementary base pairs and 2-nucleotide 3 overhangs directs RISC to the complementary target mrna induce mrna degradation only in a single, specific target sigma-aldrich.com 16

17 RNAi : short hairpin RNA (shrna) CHO-K1 cells MOI 1 Introduced into cells by : Plasmid transfection Viral vectors transduction shrna sequences : Pol III promoters (U6, H1) Selection marker : selection for the cells that stably integrate the nucleic acid into their genome System for difficult to transfect cells and long-term or controlled gene silencing An unusually compact external promoter for RNA polymerase III transcription of the human H1RNA gene Evelyne Myslinski, Jean-Christophe Amé1, Alain Krol and Philippe Carbon* UPR 9002 du CNRS Structure des Macromolécules Biologiques et Mécanismes de Reconnaissance, Institut de Biologie Moléculaire et Cellulaire, 15 rue René Descartes, Strasbourg Cedex, France and 1UPR 9003 du CNRS, Ecole Supérieure de Biotechnologie de Strasbourg, Boulevard Sébastien Brant, Illkirch, France sigma-aldrich.com 17

18 Lentiviral Gene Delivery Features Efficient infection of most cell types Dividing and non-dividing hard-to-transfect cells (primary, blood, stem cells) terminally differentiated cells (neurons, lymphocytes, etc.) Stable, heritable expression of shrna Safety Features for Mission Viral Particles sigma-aldrich.com 18

19 sigma-aldrich.com 19 Woodchuck Hepatitis Post- Transcriptional Regulatory Element (WPRE) : enhanced expression of transgenes delivered by lentivirus

20 I - Packaging into producer cell lines (HEK 293T): : production of defective particles Transduction target cell : sh expression Gag, Pol and Env available to produce and package a viral particle Only the region between the viral LTRs of the transfer vector is packaged within the viral capsid. 3rd generation system : the three plasmids share no homology, Minimizes chance of recombination events sigma-aldrich.com 20

21 shrna process sigma-aldrich.com 21

22 MISSION Products : TRC controls Untreated Cells : reference point for comparing all other samples. Empty Vectors No shrna sequence (no activation of the RNAi pathway). Non-Target shrna shrna does not target any known human or mouse gene (Activate RNAi pathway) Fluorescent proteins (TurboGFP, CFP, YFP, RFP, FP635) under the control of CMV or UbC Visual confirmation and optimization (Transduction/Transfection and MOI) shrna against reporter genes Visualize or detect knockdown in cells expressing reporters sigma-aldrich.com 22 Sigma.com/controls

23 RNAi: Detecting Knockdown mrna level : To detect the knockdown of the specific mrna transcript in the cells (compared to untreated cells): qrt-pcr, Northern Blotting Protein level : To detect the knockdown of the specific protein (compared to untreated cells) : Western Blotting, AQUA Peptides, Duolink A mrna can be reduced but with a stable protein, the protein levels may remain high Protein reduced in the absence of mrna reduction : may indicate that an sirna is mediating its effects at the translation level like a microrna. sigma-aldrich.com 23 Wistar Institute

24 RNAi: Detecting Knockdown at Protein level AQUA PEPTIDES TECHNOLOGY Synthetic Stable isotope Labeled Peptide increase (6-10 daltons) in molecular weight Mix native peptide and known amount of synthetic AQUA peptide. Digest with Trypsin Analyse by Mass spectrometry Extracted ion chromatograms are generated for the native peptide and the synthetic AQUA Peptide internal standard. Using peak ratios, the quantity of native peptide is calculated sigma-aldrich.com 24

25 RNAi: Detecting Knockdown at Protein level AQUA PEPTIDES TECHNOLOGY High sensitivity study gene silencing in low abundance proteins, or to detect very low expression of silenced genes. On any gene: an AQUA peptide can be generated using only an amino acid sequence, even when no native peptide has been isolated or when no corresponding antibody exists. AQUA quantitation measures peptide (and corresponding protein) expression. This provides a more accurate correlation to silencing efficiency when compared to quantitative PCR or Northern blotting Protein Expression Analysis in Targeted Gene RNAi Knockdown Experiments Utilizing Isotopic Labeling with 180 Water and PROTEIN-AQUA Peptides Richard J. Mehigh, Jeffrey J. Porter, Beth K. Ewing, Justin Wildsmith, Kenneth E. Heuermann, Kelly L. Foster, John G. Dapron, Steven L. Cockrill, William K. Kappel, and Graham B. I. Scott sigma-aldrich.com 25

26 RNAi: Detecting Knockdown at Protein level DUOLINK in situ PLA TECHNOLOGY Olink Bioscience (spin off Uppsala University, Sweden) A dual recognition in situ immunoassay with nucleic acid read-out sigma-aldrich.com 26

27 RNAi: Detecting Knockdown at Protein level DUOLINK in situ PLA TECHNOLOGY- Workflow overview sigma-aldrich.com 27

28 RNAi: Detecting Knockdown at Protein level DUOLINK in situ PLA TECHNOLOGY- Applications Visualize proteins under native conditions directly in the cell Protein expression Protein-protein interactions Post-translational modifications sigma-aldrich.com

29 sigma-aldrich.com RNAi tools : mirna

30 RNAi : MicroRNAs (mirnas) Non-coding endogenous small RNAs of ~22 nucleotides in length, generated by the RNase-III-type enzymes Drosha and Dicer from an endogenous transcript that contains local hairpin structure. Regulate the expression of a large number of genes namely during development. Incomplete base pairing to their target : One mirna can inhibit the translation of many different mrnas A given gene can be regulated by multiple mirnas mirnas bind to an mirna recognition element (MRE) in the 3 -untranslated region (UTR) of a target mrna Initiating its degradation or suppress its translation sigma-aldrich.com 30

31 Human mirna Mimics Library Based on mirbase version 17 : 1,902 human mirna mimics 2 non-targeting mirna human controls Functionally tested for knockdown efficiency FUNCTION - What does the mirna do? Supplement mirna function in cells having LOW endogenous levels sigma-aldrich.com 31

32 Synthetic mirna Inhibitors (human and mouse) 2'-O-methylated RNA duplexes with 2 mirna binding sites : Binds to and inhibits a specific mature mirna Excellent resistance to cellular nucleases FUNCTION - What does the mirna do? Inhibit mirna function in cells that having HIGH endogenous levels MISSION Lenti mirna Inhibitors (human and mouse) : Why? Same reasons as shrna : difficult to transfect cells and/or long term knockdown of the mirna A potent 2 -O-methylated RNA-based microrna inhibitor with unique secondary structures Nucl. Acids Res. (2012) 40 (8): e58. sigma-aldrich.com 32

33 MISSION mirna Products Isolate mirna : mirpremier microrna Isolation Kit. Convert mirna (small size, lack poly-a tail, random primers may not bind optimally) into suitable cdna templates for qpcr (polyadenylation + RT) : MystiCq microrna cdna Synthesis Mix Quantify by quantitative PCR : MystiCq microrna SYBR Green qpcr ReadyMixes sigma-aldrich.com 33

34 sigma-aldrich.com 34 Questions?