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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   

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

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

The number of different 21-mer siRNNs that can be synthesized from only the published phosphotriester groups outnumbers the number of atoms in the universe by several orders of magnitude (Meade et al., 2014). No single siRNN configuration will be the most effective for all situations. The optimal positioning, type, and number of phosphotriester groups composing a siRNN should be determined for conjugation with a particular delivery or targeting domain and delivery into target cells or tissues. In this protocol, we focus on conjugation, purification, and cellular delivery of duplexed siRNNA4 oligonucleotides with a TAT Delivery Domain (DD) peptide as described in Meade et al. (2014) (Figure 2).

  1. siRNN conjugation and purification
    1. Set up the following conjugation reaction in a 1.5 ml microcentrifuge tube in 50% ACN /50% ultrapure water (Note 2):
      1. 1x siRNN conjugation buffer
      2. 5.25 M acetic acid
      3. 50 uM siRNNA4
      4. 1 mM DD-Hynic peptide (Note 3)
    2. Incubate reaction for 1 h at RT (Note 4).
    3. After 1 h, freeze the conjugation solution in crushed dry ice.
    4. Lyophilize the frozen conjugation solution until dry (Note 5).
    5. Once dry, dissolve the lyophilized pellet in 500 μl of ultrapure water by pipetting.
    6. Load 500 μl of crude DD-siRNN conjugate into a Amicon Ultra 0.5 ml centrifugal filter unit with Ultracel-30 membrane.
    7. Centrifuge the filled filter unit for 5 min at 14,000 x g.
    8. Discard flow-through.
    9. Add 450 μl of ultrapure water to the remaining liquid in the filter unit and pipette to mix.
    10. Repeat steps A7-9 two more times.
    11. Centrifuge the filled filter unit for 10 min at 14,000 x g.
    12. Invert the filter unit into a fresh collection tube and centrifuge for 2 min at 1,000 x g to elute purified DD-siRNN conjugate.
    13. Measure the absorbance of the purified DD-siRNN conjugate at 260 nm on a UV spectrophotometer.
    14. Determine the concentration of the purified DD-siRNN conjugate using the measured absorbance at 260 nm and the duplexed siRNN molar extinction coefficient (Notes 6 and 7).
    15. Dilute the purified DD-siRNN conjugate to the desired concentration with ultrapure water. Store DD-siRNN conjugate at -20 °C (Note 8).

  2. Analysis of DD-siRNN conjugation
    1. Purchase or cast 1.5 mm 0.4% SDS 10% polyacrylamide non-denaturing gels with 10 or 15-well combs (Note 9).
    2. Aliquot 0.1 nmol of purified DD-siRNN conjugates to be analyzed in fresh microcentrifuge tubes (Note 10).
    3. Freeze samples on crushed dry ice and lyophilize until dry (Notes 11 and 12).
    4. Once dry, dissolve lyophilized samples in 5 μl of 1x 0.4% SDS non-denaturing loading buffer by pipetting (Note 13).
    5. Place polymerized gels into the gel running apparatus and fill with 0.4% SDS-PAGE running buffer. Gently remove the combs from the gels and clean out the wells by forcefully pipetting running buffer into the empty wells.
    6. Load samples dissolved in loading buffer into wells of the gel and run at 200 V constant for 30 min or until the dye front runs off the gel.
    7. Once the gel has finished running, carefully separate the glass cassette and remove the gel. Deposit the gel in a small dish for staining.
    8. Rinse the gel several times with deionized water to remove running buffer (Note 14).
    9. Add 50-100 ml of deionized water to the staining dish and incubate the gel at room temperature on a rocker for 20 min (Note 15).
    10. Discard the wash and rinse the gel with deionized water.
    11. Conduct silver stain as described in the SilverQuest Silver Staining Kit protocol under “Basic Staining Protocol” (Notes 16 and 17).
    12. Image silver-stained gels on a Gel Doc to determine degree of conjugation and purity of samples (Figure 2B).

  3. DD-siRNN cellular transduction and analysis
    1. Culture cells in desired media. For this protocol, H1299 cells stably expressing a destabilized EGFP (H1299-dGFP) were cultured in high glucose DMEM media, supplemented with 5% FBS and penicillin-streptomycin at 37 °C, 5% CO2.
    2. Rinse H1299 cells in PBS, trypsinize H1299 cells with 0.05% Trypsin-EDTA for 5 min, add growth media, and gently pipet up and down to dissociate cells from the culture dish.
    3. Centrifuge cells in bench centrifuge for 5 min at 200 x g.
    4. Discard supernatant, rinse cells in Opti-MEM, and centrifuge cells for 5 min at 200 x g.
    5. Resuspend cells in fresh Opti-MEM and dilute cells to 5 x 105 cells/ml with Opti-MEM.
    6. For each cell treatment, dilute DD-siRNN conjugates in 150 μl of fresh Opti-MEM (recommended final concentration of 50-500 nM). Transfer 150 μl of DD-siRNN dilution into a fresh well on a 24-well cell culture plate.
    7. Add 150 μl of H1299 cells (5 x 105 cells/ml) to wells containing diluted DD-siRNNs for a final concentration of 75,000 cells/well. Mix by carefully pipetting up and down several times.
    8. Incubate transductions at 37 °C, 5% CO2 for 2 h.
    9. At the end of the transduction time, aspirate off the treatment media and replace it with culture media (Note 18).
    10. After 24 h, trypsinize the transduced cells and split into three equal portions. Use two portions to seed fresh 24-well plates for later analysis of 48 and 72 h time-points.
    11. Analyze the final portion of cells on a flow cytometer to measure siRNN-induced knockdown of GFP (Figure 3A-B) (Note 19). 

Representative data



Figure 1. siRiboNucleic Neutrals (siRNNs) for cellular delivery of RNAi. siRNAs with charged phosphodiester backbones cannot cross the cell membrane unassisted. siRNNs feature bioreversible phosphotriesters which neutralize the phosphodiester backbone negative charge to allow for cellular delivery by TAT PTD delivery domain peptides and induction of RNAi responses. Source: S.F. Dowdy Lab, UCSD.



Figure 2. siRNN structure and conjugation with delivery domain (DD) peptides. A. Structures of phosphotriester groups used in this protocol. DD peptides containing hydrazine groups are conjugated to siRNNs through chemically reactive A-SATE phosphotriester group. Both the phosphotriester group and the conjugated peptide are removed by cleavage of DD-A-SATE by cytoplasmic thioesterases. B. Conjugation of DD peptides to RNN oligonucleotides containing one, two, or three A-SATE phosphotriester groups. Samples were resolved by SDS-PAGE and sliver-stained as described in this protocol. Source: S.F. Dowdy Lab, UCSD.



Figure 3. DD-siRNN cellular transduction. A. Dose curves of H1299-dGFP cells 48 h after treatment with self-delivering GFP DD-siRNNA4 or non-targeting control DD-siRNNA4. B. Histogram of H1299-dGFP cells analyzed by flow cytometry 48 h after treatment with GFP or control DD-siRNNA4 conjugates. As depicted here, the entire population undergoes an RNAi response following a successful transduction with on-target DD-siRNN conjugate. Source: S.F. Dowdy Lab, UCSD.

Notes

  1. The DD peptide (3T3S-Hy) used in this protocol was synthesized on site, but can be ordered from a variety of vendors with custom peptide synthesis services.
  2. This recipe is optimized for positively-charged DD peptide conjugation to siRNNs and may need to be adjusted to conjugate other molecules.
  3. Because there are 4 aldehyde A-SATE phosphotriester groups per molecule of siRNNA4 this results in a 5:1 ratio of DD peptide to A-SATE.
  4. For reaction volumes greater than 250 μl it is advantageous to conduct this reaction on a rotisserie.
  5. It is important that the conjugation solution be lyophilized until fully dry as residual acetic acid and ACN can distort the centrifugal filter membrane, preventing purification of DD-siRNN conjugates. The amount of time required to lyophilize a sample is dependent on the equipment used. A lyophilization rate of 50 μl/h can be expected using the equipment listed in this protocol with a vacuum pressure of 200 mTorr and a condenser temperature of -80 °C.
  6. Use the Beer-Lambert Law to calculate the concentration of the purified DD-siRNN conjugate. The Beer-Lambert Law: A=ϵlc where A is the measure of absorbance (at 260 nm in this case), ϵ is the molar extinction coefficient, l is the path length (1 cm for most spectrophotometers), and c is the concentration of the solution.
  7. The molar extinction coefficient for a duplexed oligonucleotide should be empirically determined. 292,500 (M-1 cm-1) is the molar extinction coefficient for the GFP siRNN used in this protocol.
  8. 20 μM is a useful concentration to dilute DD-siRNN conjugates for 24-well plate treatments.
  9. If casting your own gels make 0.4% SDS non-denaturing polyacrylamide gel solution in a 50 ml conical tube. Add 200 μl of 10% ammonium persulfate and 800 μl of 10% SDS solution to the gel solution. Mix by inverting the tube several times. Add 20 μl of TEMED to the gel solution and invert the tube several times to mix. Cast two 1.5 mm gels with the gel solution and glass plates. Carefully insert gel combs without introducing or trapping any air bubbles. Let gels sit at room temperature for 20 min to ensure complete polymerization before use.
  10. If DD-siRNN conjugates were diluted to 20 μM, 5 μl (0.1 nmol) should be aliquoted for analysis.
  11. Dry ice is extremely cold (-78.5 °C). Always handle dry ice with care and wear appropriate personal protective equipment (eye protection and insulated gloves).
  12. Lyophilization time will be dependent on the equipment and volume, but 30 min should be sufficient to fully dry 5 μl of DD-siRNN conjugate dissolved in water.
  13. Do not heat DD-siRNN conjugates as this may result in melting of the siRNN duplex. Once conjugated to a DD peptide, single-stranded RNNs (ssRNNs) will not readily duplex.
  14. A 12 x 10 x 3 cm polypropylene dish was used for staining in this protocol.
  15. This step decreases background staining during the silver stain by washing some SDS out of the gel.
  16. The SilverQuest Silver Staining Kit manual recommends using 100 ml for all solutions, but 50 ml may be used instead as long as the staining dish is small enough for 50 ml to completely cover the gel.
  17. If 0.1 nmol of DD-siRNN conjugate is loaded, 1-5 min of silver stain development time should be sufficient to visualize the conjugate.
  18. Under these conditions, H1299 cells will adhere to the plate after 2 h. If the cells do not adhere sufficiently for complete aspiration of the transduction media, add 500 μl of culture media, incubate the plate until the cells adhere, and then aspirate all of the media.
  19. For this protocol, H1299-dGFP cells were analyzed on a LSR II flow cytometer. dGFP was measured using a 488 nm laser and a 530/30 emission filter. 5 x 104 events were collected and gated according to forward scatter (FSC-A) and side scatter (SSC-A).
  20. Abbreviations used in this protocol: siRNA = short interfering RNA, PTD = peptide transduction domain, siRNN = siRiboNucleic Neutral, DMB = dimethyl butyl, SATE = S-acyl-2-thioethyl, HyNic = 6-hydrazinonicotinamide.

Recipes

  1. 5x siRNN conjugation buffer (1 ml)
    500 μl acetonitrile
    315.5 μl ultrapure water
    9.5 μl 10.5 M aniline (100 mM final concentration)
    125 μl of 200 mM HEPES (pH 5.5) (25 mM final concentration)
    50 μl 5 M NaCl (250 mM final concentration)
  2. 0.4% SDS non-denaturing polyacrylamide gel solution
    12.2 ml of ultrapure water
    5 ml 40% acrylamide/bis solution
    2 ml 10x TBE
  3. 0.4% SDS-PAGE running buffer (1 L)
    40 ml of 10% SDS solution
    100 ml of 10x TBE
    860 ml deionized water
  4. 1x 0.4% SDS non-denaturing loading buffer (1 ml)
    40 μl 10% SDS solution
    250 μl glycerol
    2 mg Orange G
    Add ultrapure water to 1 ml

Acknowledgments

The protocol described herein was developed and utilized previously in Meade et al. (2014). A. S. H. was supported by a T32 Cancer Biology Training grant (NCI) and by a Blasker Award from The San Diego Foundation. This work was supported by the W.M. Keck Foundation (S. F. D.), the Department of Defense (S. F. D.), SCOR grant from the Leukemia & Lymphoma Society (S. F. D.), the Pardee Foundation (S. F. D.), a grant from an anonymous donor (S. F. D.) and the Howard Hughes Medical Institute (S. F. D.).
The authors declare competing financial interests. S. F. D. and K.G. (UCSD) have filed patents on this work that were licensed by Solstice Biologics, Inc. (San Diego). S. F. D. is a cofounder of Solstice Biologics. S. F. D. is a board director of Solstice Biologics.

References

  1. Bumcrot, D., Manoharan, M., Koteliansky, V. and Sah, D. W. (2006). RNAi therapeutics: a potential new class of pharmaceutical drugs. Nat Chem Biol 2(12): 711-719.
  2. Farkhani, S. M., Valizadeh, A., Karami, H., Mohammadi, S., Sohrabi, N. and Badrzadeh, F. (2014). Cell penetrating peptides: efficient vectors for delivery of nanoparticles, nanocarriers, therapeutic and diagnostic molecules. Peptides 57: 78-94.
  3. Meade, B. R. and Dowdy, S. F. (2007). Exogenous siRNA delivery using peptide transduction domains/cell penetrating peptides. Adv Drug Deliv Rev 59(2-3): 134-140.
  4. Meade, B. R., Gogoi, K., Hamil, A. S., Palm-Apergi, C., van den Berg, A., Hagopian, J. C., Springer, A. D., Eguchi, A., Kacsinta, A. D., Dowdy, C. F., Presente, A., Lonn, P., Kaulich, M., Yoshioka, N., Gros, E., Cui, X. S. and Dowdy, S. F. (2014). Efficient delivery of RNAi prodrugs containing reversible charge-neutralizing phosphotriester backbone modifications. Nat Biotechnol 32(12): 1256-1261.

简介

尽管由于它们的大小(〜15kDa)和高负电荷(Bumcrot等人,2006),短干扰RNA(siRNA)诱导的RNAi反应保持作为治疗模式的巨大希望,siRNA没有生物利用度,需要递送剂进入细胞(图1)。 TAT肽转导结构域(PTD)已经被开发为介导大分子治疗剂的细胞递送的药剂,否则其缺乏生物利用度,使其成为siRNA递送的诱人候选物(Farkhani等人,2014)。不幸的是,当缀合到TAT PTD时,siRNA上40个负磷酸二酯主链电荷的存在中和了导致聚集和差的细胞递送的阳离子PTD(Meade和Dowdy,2007)。鉴于此,我们合成了称为siRibo核中性的中性RNAi触发物,用于与TAT PTD缀合(Meade等人,2014)。简言之,带负电荷的磷酸二酯主链通过具有生物可逆磷酸三酯保护基团的合成来中和,其通过细胞质限制性硫酯酶的作用特异性地转化为细胞内的带电磷酸二酯键,产生可诱导RNAi应答的野生型siRNA。在这里我们描述了与TAT PTD递送结构域(DD)HyNic肽的siRNN寡核苷酸的缀合和细胞递送。

关键字:siRNA, siRNN, 磷酸三酯, 肽转导域, 寡核苷酸共轭

材料和试剂

  1. 1.5ml微量离心管(聚丙烯,热原,RNase,无DNA酶)
  2. 带有Ultracel-30膜的Amicon Ultra 0.5ml离心过滤单元(Merck Millipore Corporation,目录号:UFC503024)
  3. 50ml锥形离心管(VWR International,目录号:21008-714)
  4. 24孔细胞培养板,平底,TC处理(Genesse Scientific,目录号:25-107)
  5. H1299细胞(ATCC,目录号:CRL-5803) 注意:在该方案中使用组成型表达不稳定增强型绿色荧光蛋白(dGFP)的H1299细胞,通过流式细胞术快速测定RNAi应答。
  6. GFP和非靶向siRNA(RNN亚磷酰胺和寡核苷酸合成描述于Meade等人,2014),RNN序列(5'至3'):
    1. GFP过客链:C CACU ACCUGAGC ACC A CAGU
    2. GFP引导链:CU GGGU GCU CAGGU AGU GGU S T
    3. 非靶向过客链:U亚基GAGA A GAUCCUCA亚基UAA A亚基AGAU亚基> T
    4. 非靶向引导链:U S UUUA S UGA S S GGAUC SubSUU S sub > CAU S T
    5. 关键词:下标D =二甲基 - 丁基磷酸三酯组,下标A ?=醛A-SATE磷酸三酯组,下标S = t Bu-SATE 磷酸三酯组
  7. UltraPure DNase/RNase-free蒸馏水(Life Technologies,Invitrogen,目录号:10977023) 注意:目前,它是"Thermo Fisher Scientific,Invitrogen TM ,目录号:10977023"。
  8. TAT递送结构域(DD)Hynic肽,3T3S-Hy [TAT = RKKRRQRRR,3T3S-Hy序列:HyNic-GG-(TAT)-PEG18-(TAT)-PEG18-(TAT)]
  9. 乙腈(ACN),无水(Glen Research,目录号:40-4050-50)
  10. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653)
  11. HEPES(AmericanBio,目录号:AB00892)
  12. 苯胺(Alfa Aesar,目录号:A14443)
  13. 乙酸,Glacial(Thermo Fisher Scientific,目录号:MAX00739)
  14. 干冰
  15. 40%丙烯酰胺/双溶液(19:1)(Bio-Rad Laboratories,目录号:161-0144)
  16. 10x Tris-硼酸盐-EDTA(TBE)缓冲液(Mediatech,目录号:46-011-CM)
  17. 过硫酸铵(VWR International,目录号:EM-2300)
  18. 10%(w/v)十二烷基硫酸钠(SDS)溶液(Bio-Rad Laboratories,目录号:161-0416)
  19. N,N,N',N'-四甲基乙二胺(TEMED)(VWR International,目录号:EM-8920)
  20. 去离子水
  21. Orange G(Sigma-Aldrich,目录号:O-3756)
  22. 甘油(Sigma-Aldrich,目录号:G5516)
  23. SilverQuest银染试剂盒(Life Technologies,Novex?,目录号:LC6070)
    注意:目前,"Thermo Fisher Scientific,Invitrogen TM ,目录号:LC6070"。
  24. DMEM,高葡萄糖(Life Technologies,Gibco,目录号:11965-092) 注意:目前,它是"Thermo Fisher Scientific,Gibco TM ,目录号:11965-092"。
  25. 胎牛血清(FBS),热灭活(Omega Scientific,目录号:FB-02)
  26. 青霉素 - 链霉素(10,000U/ml)(Life Technologies,Gibco,目录号:15140-122)
    注意:目前,它是"Thermo Fisher Scientific,Gibco TM ,目录号:15140-122"。
  27. 磷酸盐缓冲盐水(Thermo Fisher Scientific,目录号:BP665-1)
  28. Opti-MEM减少的血清培养基(Life Technologies,Gibco,目录号:31985-070) 注意:目前,它是"Thermo Fisher Scientific,Gibco TM ,目录号:31985-070"。
  29. 0.05%胰蛋白酶-EDTA,酚红(Life Technologies,Gibco,目录号:25300-054) 注意:目前,"Thermo Fisher Scientific,Gibco TM ,目录号:25300-054"。
  30. 5x siRNN共轭缓冲液(见配方)
  31. 0.4%SDS非变性聚丙烯酰胺凝胶溶液(见配方)
  32. 0.4%SDS-PAGE电泳缓冲液(见配方)
  33. 1x 0.4%SDS非变性上样缓冲液(参见配方)

设备

  1. Rotisserie(Barnstead International,型号:Labquake 4001100)
  2. 冻干器(SP Scientific,VirTis,型号:Freezemobile 25EL)
  3. 微量离心机(Eppendorf,型号:5424)
  4. UV分光光度计(能够测量260nm处的吸光度)
  5. 聚丙烯酰胺凝胶浇铸台和带有1.5mm间隔物的玻璃(Bio-Rad Laboratories)
  6. 凝胶流动装置(Bio-Rad Laboratories,型号:Mini-PROTEAN 3 Cell)
  7. 凝胶染色皿
  8. Gel Doc(Bio-Rad Laboratories,型号:Molecular Imager Gel Doc XR + System)
  9. 台式离心机(Beckman Coulter,型号:Allegra X-15R)
  10. 流式细胞仪(BD Sciences,型号:LSRII)

程序

可以仅由公开的磷酸三酯组合成的不同21聚体siRNN的数量比宇宙中原子的数量多几个数量级(Meade等人,2014)。没有单个siRNN配置将是最有效的所有情况。应当确定构成siRNN的磷酸三酯组的最佳定位,类型和数目,用于??与特定递送或靶向结构域缀合并递送到靶细胞或组织中。在该方案中,我们集中于如Meade等人所述的具有TAT递送结构域(DD)肽的双链体siRNN寡核苷酸的缀合,纯化和细胞递送。 2014)(图2)。

  1. siRNN共轭和纯化
    1. 在1.5ml微量离心管中在50%ACN/50%超纯水(注2)中设置以下缀合反应:
      1. 1x siRNN共轭缓冲区
      2. 5.25M乙酸
      3. 50 uM siRNN A4
      4. 1 mM DD-Hynic肽(注3)
    2. 在室温下孵育反应1小时(注4)。
    3. 1小时后,将共轭溶液在碎干冰中冷冻
    4. 冻干冻结的共轭溶液直至干燥(注释5)
    5. 一旦干燥,通过移液将冻干颗粒溶解在500μl超纯水中
    6. 将500μl粗DD-siRNN缀合物装入具有Ultracel-30膜的Amicon Ultra 0.5ml离心过滤装置中。
    7. 将填充的过滤器单元以14,000×g离心5分钟。
    8. 丢弃流通。
    9. 在过滤器单元和移液器中的剩余液体中加入450μl超纯水混合
    10. 重复步骤A7-9两次以上。
    11. 将填充的过滤器单元在14,000×g离心10分钟。
    12. 将过滤器单元倒入新鲜的收集管和离心机中 在1,000×g下洗脱2分钟以洗脱纯化的DD-siRNN缀合物
    13. 在紫外分光光度计上测量纯化的DD-siRNN缀合物在260nm的吸光度
    14. 测定纯化的DD-siRNN缀合物的浓度 使用在260nm测量的吸光度和双重的siRNN摩尔 消光系数(注6和7)。
    15. 稀释纯化 DD-siRNN缀合物与超纯水混合至所需浓度。 将DD-siRNN缀合物储存在-20°C(注8)。

  2. DD-siRNN共轭分析
    1. 购买或浇铸1.5 mm 0.4%SDS 10%聚丙烯酰胺非变性凝胶,带10或??15孔梳子(注9)。
    2. 等分0.1 nmol纯化的DD-siRNN共轭物,在新鲜的微量离心管中进行分析(注10)。
    3. 在粉碎的干冰上冻结样品,冻干至干燥(注释11和12)
    4. 一旦干燥,通过移液(注13)将冻干样品溶解在5μl的1x 0.4%SDS非变性加载缓冲液中。
    5. 将聚合凝胶放入凝胶运行装置中并填充 0.4%SDS-PAGE电泳缓冲液。轻轻地从凝胶和 通过强力吸取运行缓冲液清除孔 空井
    6. 将溶解在加样缓冲液中的样品装载到孔中 的凝胶并在200V恒定下运行30分钟或直到染料前沿 跑掉凝胶。
    7. 一旦凝胶完成运行,仔细 分离玻璃盒并除去凝胶。将凝胶沉积在 小盘染色。
    8. 用去离子水冲洗凝胶数次以去除运行缓冲液(注14)
    9. 向染色皿中加入50-100ml去离子水并孵育 凝胶在室温下摇动20分钟(注15)
    10. 弃去洗涤液,用去离子水冲洗凝胶。
    11. 按照SilverQuest Silver中所述进行银色染色 "基本染色方案"下的染色试剂盒协议(注16和17)
    12. 在凝胶上图像银染色的凝胶以确定样品的缀合度和纯度(图2B)。

  3. DD-siRNN细胞转导和分析
    1. 在所需培养基中培养细胞。对于该方案,H1299细胞稳定 ?表达不稳定的EGFP(H1299-dGFP) 葡萄糖DMEM培养基,补充有5%FBS和青霉素 - 链霉素 ?在37℃,5%CO 2下
    2. 冲洗H1299细胞在PBS,胰蛋白酶H1299 细胞与0.05%胰蛋白酶-EDTA 5分钟,添加生长培养基,并温和 用移液管向上和向下移动以从培养皿中分离细胞
    3. 在台式离心机中以200×g离心细胞5分钟。
    4. 弃去上清液,在Opti-MEM中漂洗细胞,并在200×g离心细胞5分钟。
    5. 重悬细胞在新鲜的Opti-MEM中,并用Opti-MEM稀释细胞至5×10 5个细胞/ml。
    6. 对于每个细胞处理,稀释DD-siRNN缀合物在150μl 新鲜Opti-MEM(推荐终浓度为50-500 nM)。转让 将150μlDD-siRNN稀释液置于24孔细胞培养物的新鲜孔中 板
    7. 向孔中加入150μlH1299细胞(5×10 5个细胞/ml) 含有稀释的DD-siRNNs,最终浓度为75,000 细胞/孔。通过小心地上下吹打数次混合。
    8. 在37℃,5%CO 2孵育转导2小时
    9. 在转导时间结束时,吸出治疗介质并用培养基更换(注18)
    10. 24小时后,胰蛋白酶处理转导的细胞并分成三份 相等部分。使用两部分种子新鲜24孔板以后 分析48和72小时的时间点
    11. 在流式细胞仪上分析细胞的最终部分以测量siRNN诱导的GFP的敲低(图3A-B)(注19)。
      014)。没有单个siRNN配置将是最有效的 情况。磷酸三酯的最佳定位,类型和数量 ?应确定组成siRNN的基团以与a缀合 特定的递送或靶向结构域和递送到靶细胞中 或组织。在这个协议,我们专注于共轭,纯化,和 ?用TAT细胞递送双链体siRNNA4寡核苷酸 交付域(DD)肽,如Meade等人(2014年) ?2)。

代表数据



图1.用于RNAi的细胞递送的siRibo核中性(siRNNs)。带有带电荷的磷酸二酯骨架的siRNA不能穿过细胞膜而无帮助。 siRNNs特征在于生物可逆磷酸三酯,其中和磷酸二酯骨架负电荷以允许通过TAT PTD递送结构域肽的细胞递送和诱导RNAi应答。资料来源:S.F. Dowdy实验室,UCSD。



图2.siRNN结构和与递送结构域(DD)肽的缀合。A.本方案中使用的磷酸三酯基团的结构。含有肼基的DD肽通过化学反应性A-SATE磷酸三酯基团与siRNNs缀合。通过细胞质硫酯酶切割DD-A-SATE除去磷酸三酯基团和缀合肽。 B.DD肽与含有一个,两个或三个A-SATE磷酸三酯基团的RNN寡核苷酸的缀合。通过SDS-PAGE分离样品,并如本方案中所述进行条染色。资料来源:S.F. Dowdy实验室,UCSD。



图3.DD-siRNN细胞转导 A. H1299-dGFP细胞在用自体递送GFP DD-siRNNA4或非靶向对照DD-siRNNA4处理48小时后的剂量曲线。 B.用GFP或对照DD-siRNNA4缀合物处理48小时后,通过流式细胞术分析H1299-dGFP细胞的直方图。如本文所述,在用靶向DD-siRNN缀合物成功转导后,整个群体经历RNAi应答。资料来源:S.F. Dowdy实验室,UCSD。

笔记

  1. 本方案中使用的DD肽(3T3S-Hy)是现场合成的,但可以从具有定制肽合成服务的各种供应商订购。
  2. 该配方针对带正电荷的DD肽与siRNN的缀合而优化,并且可能需要调节以与其他分子缀合。
  3. 因为每个siRNN A4分子中有4个醛A-SATE磷酸三酯基团,这导致DD肽与A-SATE的比例为5:1。
  4. 对于大于250μl的反应体积,在旋转式烤架上进行该反应是有利的
  5. 重要的是将缀合溶液冻干直至完全干燥,因为残留的乙酸和ACN可以使离心过滤膜变形,从而防止DD-siRNN缀合物的纯化。冻干样品所需的时间取决于所使用的设备。使用本方案中列出的设备,在真空压力为200mTorr和冷凝器温度为-80℃下,可以预期50μl/h的冻干速率。
  6. 使用Beer-Lambert定律计算纯化的DD-siRNN缀合物的浓度。 Beer-Lambert定律:A =εlc其中A是吸光度(在这种情况下在260nm)的测量,ε是摩尔消光系数,l是路径长度(对于大多数分光光度计为1cm),c是浓度的溶液。
  7. 双链寡核苷酸的摩尔消光系数应该根据经验确定。 292,500(M <= sup-1 -1 )是本方案中使用的GFP siRNN的摩尔消光系数。
  8. 20μM是用于稀释用于24孔板处理的DD-siRNN缀合物的有用浓度。
  9. 如果铸造自己的凝胶,在50毫升锥形管中制备0.4%SDS非变性聚丙烯酰胺凝胶溶液。向凝胶溶液中加入200μl的10%过硫酸铵和800μl的10%SDS溶液。混合通过倒置管几次。添加20微升TEMED到凝胶溶液,倒置管数次混合。用凝胶溶液和玻璃板浇铸两个1.5mm凝胶。小心地插入凝胶梳,而不引入或捕获任何气泡。让凝胶在室温下放置20分钟,以确保在使用前完全聚合
  10. 如果DD-siRNN缀合物稀释至20μM,应分装5μl(0.1 nmol)用于分析。
  11. 干冰非常冷(-78.5℃)。始终小心处理干冰,并佩戴适当的个人防护装备(护目镜和绝缘手套)。
  12. 冻干时间将取决于设备和体积,但30分钟应足以完全干燥溶解在水中的5μlDD-siRNN缀合物。
  13. 不要加热DD-siRNN缀合物,因为这可能导致siRNN双链体熔化。一旦与DD肽缀合,单链RNN(ssRNN)将不容易复制
  14. 在该方案中使用12×10×3cm的聚丙烯培养皿进行染色
  15. 此步骤通过从凝胶中洗去一些SDS而减少银染期间的背景染色
  16. SilverQuest银染试剂盒手册建议所有溶液使用100毫升,但可以使用50毫升,只要染色皿足够小,50毫升完全覆盖凝胶。
  17. 如果加载0.1nmol的DD-siRNN缀合物,则1-5分钟的银染色显影时间应足以显现缀合物。
  18. 在这些条件下,H1299细胞在2小时后将粘附在平板上。如果细胞不足以完全吸引转导培养基,加入500μl培养基,孵育板直至细胞粘附,然后吸出所有培养基。
  19. 对于该方案,在LSR II流式细胞仪上分析H1299-dGFP细胞。使用488nm激光和530/30发射滤光片测量dGFP。根据前向散射(FSC-A)和侧向散射(SSC-A)收集和门控5×10 4次事件。
  20. 本方案中使用的缩写:siRNA =短干扰RNA,PTD =肽转导结构域,siRNN = siRibo核酸中性,DMB =二甲基丁基,SATE = S-酰基-2-硫代乙基,HyNic = 6-肼基烟酰胺。

食谱

  1. 5xsiRNN偶联缓冲液(1ml) 500μl乙腈
    315.5μl超纯水
    9.5μl10.5M苯胺(100mM终浓度)
    125μl200mM HEPES(pH5.5)(25mM终浓度) 50μl5M NaCl(250mM终浓度)
  2. 0.4%SDS非变性聚丙烯酰胺凝胶溶液 12.2ml超纯水
    5ml 40%丙烯酰胺/双溶液
    2 ml 10x TBE
  3. 0.4%SDS-PAGE电泳缓冲液(1L) 40ml 10%SDS溶液
    100ml 10×TBE
    860ml去离子水
  4. 1x 0.4%SDS非变性上样缓冲液(1ml) 40μl10%SDS溶液
    250微升甘油 2 mg橙色G
    将超纯水加入1 ml

致谢

本文所述的方案是先前在Meade等人(2014)中开发和利用的。 A.S.H。得到T32癌症生物学培训资助(NCI)和来自圣地亚哥基金会的Blasker奖的支持。这项工作得到了W.M. Keck基金会(S.F.D.),国防部(S.F.D.),来自白血病&淋巴瘤协会(S.F.D.),Pardee基金会(S.F.D.),来自匿名捐赠者(S.F.D.)和霍华德休斯医学研究所(S.F.D.)的赠款。
作者声明竞争的经济利益。 S.F.D和K.G. (UCSD)已经提交了由Solstice Biologics,Inc.(圣地亚哥)许可的这项工作的专利。 S.F.D.是Solstice Biologics的共同创办人。 S.F.D.是Solstice Biologics的董事会主席。

参考文献

  1. Bumcrot,D.,Manoharan,M.,Koteliansky,V.and Sah,D.W。(2006)。 RNAi疗法:一种潜在的新型药物。 Nat Chem Biol 2(12):711-719。
  2. Farkhani,S.M.,Valizadeh,A.,Karami,H.,Mohammadi,S.,Sohrabi,N.and Badrzadeh,F。 细胞穿透肽:用于递送纳米颗粒,纳米载体,治疗和诊断分子的高效载体。 Peptides 57:78-94
  3. Meade,B.R。和Dowdy,S.F。(2007)。 使用肽转导结构域/细胞穿透肽的外源性siRNA递送。 Deliv Rev 59(2-3):134-140
  4. Meade,BR,Gogoi,K.,Hamil,AS,Palm-Apergi,C.,van den Berg,A.,Hagopian,JC,Springer,AD,Eguchi,A.,Kacsinta,AD,Dowdy,CF,Presente, A.,Lonn,P.,Kaulich,M.,Yoshioka,N.,Gros,E.,Cui,XS和Dowdy,SF(2014)。 含有可逆电荷中和磷酸三酯骨架修饰的RNAi前药的有效递送。 Nat Biotechnol 32(12):1256-1261。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
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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