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Conjugation of Duplexed siRNN Oligonucleotides with DD-HyNic Peptides for Cellular Delivery of RNAi Triggers

双 siRNN 寡核苷酸与DD-HyNic 肽接合用于RNAi触发的细胞传递分析

AH Alexander S. Hamil
KG Khirud Gogoi
SD Steven F. Dowdy

发布: 2016年04月05日第6卷第7期 DOI: 10.21769/BioProtoc.1782 浏览次数: 8679

评审: Arsalan DaudiVikash VermaAnonymous reviewer(s)

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Abstract

Despite the great promise that short interfering RNA (siRNA) induced RNAi responses hold as a therapeutic modality, due to their size (~15 kDa) and high negative charge (Bumcrot et al., 2006), siRNAs have no bioavailability and require a delivery agent to enter cells (Figure 1). TAT peptide transduction domain (PTD) has been developed as an agent that mediates cellular delivery of macromolecular therapeutics that otherwise lack bioavailability, making it a tantalizing candidate for siRNA delivery (Farkhani et al., 2014). Unfortunately, when conjugated to TAT PTD, the presence of 40 negative phosphodiester backbone charges on siRNA neutralizes the cationic PTD resulting in aggregation and poor cellular delivery (Meade and Dowdy, 2007). In light of this, we synthesized a neutral RNAi trigger, termed siRiboNucleic Neutrals, for conjugation to TAT PTD (Meade et al., 2014). In brief, the negatively charged phosphodiester backbone was neutralized by synthesis with bio-reversible phosphotriester protecting groups which are specifically converted into charged phosphodiester bonds inside of cells by the action of cytoplasmic restricted thioesterases resulting in a wild type siRNA that can induce RNAi responses. Here we describe the conjugation and cellular delivery of siRNN oligonucleotides with TAT PTD delivery domain (DD) HyNic peptides.

Keywords: siRNA (siRNA) search siRNN (siRNN) search Phosphotriester (磷酸三酯) search Peptide Transduction Domain (肽转导域) search Oligonucleotide conjugation (寡核苷酸共轭) search

Materials and Reagents

  1. 1.5 ml microcentrifuge tubes (Polypropylene, Pyrogen, RNase, DNase-free)
  2. Amicon Ultra 0.5 ml centrifugal filter unit with Ultracel-30 membrane (Merck Millipore Corporation, catalog number: UFC503024 )
  3. 50 ml conical centrifuge tubes (VWR International, catalog number: 21008-714 )
  4. 24-well cell culture plates, flat bottom, TC treated (Genesee Scientific, catalog number: 25-107 )
  5. H1299 cells (ATCC, catalog number: CRL-5803 )
    Note: H1299 cells constitutively expressing destabilized enhanced green fluorescent protein (dGFP) are used in this protocol for rapid assaying of RNAi responses by flow cytometry.
  6. GFP and non-targeting siRNNA4 (RNN phosphoramidite and oligonucleotide synthesis is described in Meade et al., 2014), RNN sequences (5’ to 3’):
    1. GFP passenger strand: CDCACUAACCUGAGCAACCACAGUAT
    2. GFP guide strand: CUSGGGUSGCUSCAGGUSAGUSGGUST
    3. Non-targeting passenger strand: UDGAGAAGAUCCUCAAUAAAAGAUAT
    4. Non-targeting guide strand: UCSUUUASUGASGGAUCSUCUSCAUST
    5. Key: subscript D = dimethyl-butyl phosphotriester group, subscript A = aldehyde A-SATE phosphotriester group, subscript S = tBu-SATE phosphotriester group
  7. UltraPure DNase/RNase-free distilled water (Life Technologies, Invitrogen, catalog number: 10977023 )
    Note: Currently, it is “Thermo Fisher Scientific, InvitrogenTM, catalog number: 10977023”.
  8. TAT Delivery Domain (DD) Hynic peptide, 3T3S-Hy [TAT = RKKRRQRRR, 3T3S-Hy sequence: HyNic-GG-(TAT)-PEG18-(TAT)-PEG18-(TAT)] (Note 1)
  9. Acetonitrile (ACN), Anhydrous (Glen Research, catalog number: 40-4050-50 )
  10. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
  11. HEPES (AmericanBio, catalog number: AB00892 )
  12. Aniline (Alfa Aesar, catalog number: A14443 )
  13. Acetic Acid, Glacial (Thermo Fisher Scientific, catalog number: MAX00739 )
  14. Dry ice
  15. 40% acrylamide/bis solution (19:1) (Bio-Rad Laboratories, catalog number: 161-0144 )
  16. 10x Tris-Borate-EDTA (TBE) Buffer (Mediatech, catalog number: 46-011-CM )
  17. Ammonium persulfate (VWR International, catalog number: EM-2300 )
  18. 10% (w/v) sodium dodecyl sulfate (SDS) solution (Bio-Rad Laboratories, catalog number: 161-0416 )
  19. N, N, N’, N’-Tetramethylethylenediamine (TEMED) (VWR International, catalog number: EM-8920 )
  20. Deionized water
  21. Orange G (Sigma-Aldrich, catalog number: O-3756 )
  22. Glycerol (Sigma-Aldrich, catalog number: G5516 )
  23. SilverQuest Silver Staining Kit (Life Technologies, Novex™, catalog number: LC6070 )
    Note: Currently, it is “Thermo Fisher Scientific, InvitrogenTM, catalog number: LC6070”.
  24. DMEM, high glucose (Life Technologies, Gibco, catalog number: 11965-092 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 11965-092”.
  25. Fetal Bovine Serum (FBS), heat inactivated (Omega Scientific, catalog number: FB-02 )
  26. Penicillin-Streptomycin (10,000 U/ml) (Life Technologies, Gibco, catalog number: 15140-122 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 15140-122”.
  27. Phosphate buffered saline (Thermo Fisher Scientific, catalog number: BP665-1 )
  28. Opti-MEM reduced serum medium (Life Technologies, Gibco, catalog number: 31985-070 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 31985-070”.
  29. 0.05% Trypsin-EDTA, phenol red (Life Technologies, Gibco, catalog number: 25300-054 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 25300-054”.
  30. 5x siRNN conjugation buffer (see Recipes)
  31. 0.4% SDS non-denaturing polyacrylamide gel solution (see Recipes)
  32. 0.4% SDS-PAGE running buffer (see Recipes)
  33. 1x 0.4% SDS non-denaturing loading buffer (see Recipes)

Equipment

  1. Rotisserie (Barnstead International, model: Labquake 4001100 )
  2. Lyophilizer (SP Scientific, VirTis, model: Freezemobile 25EL )
  3. Microcentrifuge (Eppendorf, model: 5424 )
  4. UV Spectrophotometer (capable of measuring absorbance at 260 nm)
  5. Polyacrylamide gel casting stand and glass with 1.5 mm spacers (Bio-Rad Laboratories)
  6. Gel running apparatus (Bio-Rad Laboratories, model: Mini-PROTEAN 3 Cell )
  7. Gel staining dish
  8. Gel Doc (Bio-Rad Laboratories, model: Molecular Imager Gel Doc XR+ System )
  9. Benchtop centrifuge (Beckman Coulter, model: Allegra X-15R )
  10. Flow cytometer (BD Sciences, model: LSRII )

Procedure

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文章信息

版权信息

© 2016 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Hamil, A. S., Gogoi, K. and Dowdy, S. F. (2016). Conjugation of Duplexed siRNN Oligonucleotides with DD-HyNic Peptides for Cellular Delivery of RNAi Triggers. Bio-protocol 6(7): e1782. DOI: 10.21769/BioProtoc.1782.

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分类

分子生物学 > RNA > RNA 干扰

分子生物学 > RNA > RNA 合成

生物化学 > RNA > RNA结构

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