Synthesis and Evaluation of Caged siRNA with Terminal Single Vitamin E Modification

Jiali Yang1, Lijia Yu1, Liangliang Zhang1, Xingsu Long1, Yuzhuo Ji1, Xinjing Tang1

1 State Key Laboratory of Natural and Biomimetic Drugs, Center of Noncoding RNA Medicine, The School of Pharmaceutical Sciences, Peking University, Beijing
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 16.6
DOI:  10.1002/cpnc.16
Online Posting Date:  December, 2016
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Abstract

RNA‐induced gene silencing has been widely applied as a powerful research tool in drug development due to its sequence‐specific degradation of target mRNA. Conditional regulation of gene functions with small interfering RNAs (siRNAs) is highly useful, especially when specific gene expression regulation with spatiotemporal resolution and amplitude is desired. Here, the synthesis of a series of new caged siRNAs with vitamin E (vitE) modification and/or a single photolabile linker at the 5′ terminal is described. Their capability of photolysis was investigated by PAGE gel analysis. Then, a dual reporter firefly/renilla luciferase assay with siQuant vectors and GFP/RFP reporter genes was applied to show the effect of vitE‐modified caged and non‐caged siRNAs on gene expression. The intracellular distribution and cellular uptake pathways of caged siRNAs are also discussed. © 2016 by John Wiley & Sons, Inc.

Keywords: caged siRNA; photoactivation; RNAi; photochemical biology; vitamin E modification

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesize Photolinker (PL) Phosphoramidite and Vitamin E Phophoramidite
  • Basic Protocol 2: Synthesis, Purification, and Characterization of Caged RNA Oligoribonucleotides
  • Basic Protocol 3: Photochemical Regulation of Gene Expression Using Caged siRNAs for Reporter Genes (GFP/RFP and Firefly/Renilla Luciferase)
  • Basic Protocol 4: Intracellular Distribution and Cellular Uptake Pathways
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Synthesize Photolinker (PL) Phosphoramidite and Vitamin E Phophoramidite

  Materials
  • 2′‐Nitroacetophenone (97%, Alfa Aesar)
  • Copper II bromide (98% CuBr 2, Sigma‐Aldrich)
  • Ethyl acetate (analytical grade)
  • Chloroform (analytical grade)
  • Bromine (Aladdin)
  • Saturated brine
  • Sodium sulfate (Na 2SO 4, Aladdin), anhydrous
  • 1,4‐Dioxane (analytical grade)
  • Sodium borohydride (NaBH 4, Macklin)
  • Methanol
  • Thin‐layer chromatography (TLC) plates (Silica gel 60 F, Merck)
  • Petroleum ether (analytical grade)
  • 5%sodium hydroxide (NaOH, Aladdin)
  • Silica gel (Merck, type 9385, 230 to 400 mesh)
  • Triethylamine (analytical grade)
  • Potassium carbonate (K 2CO 3, Aladdin)
  • Hydrochloric acid (37%)
  • Dichloromethane (analytical‐grade CH 2Cl 2)
  • 4,4′‐Dimethoxytriphenylmethyl chloride (DMTr·Cl, Macklin)
  • Ca 2H 2
  • Nitrogen or argon gas (N 2 or Ar, respectively), dry
  • 2‐Cyanoethyl N,N,N′,N′‐tetraisopropylphosphordiamidite (Aktis)
  • CDCl 3 (J&K)
  • Vitamin E (vitE 96%,TCI)
  • 2‐Cyanoethyl N,N‐diisopropylchlorophosphoramidite (> 97%, Aldrich)
  • Tetrazole (Macklin)
  • 100‐ and 250‐mL three‐neck, round‐bottom flasks
  • Magnetic stir bars and plate
  • Allihn condensers
  • Rotary evaporator
  • 25‐, 50‐, and 100‐mL round‐bottom flasks
  • 250‐mL separating funnel
  • Büchner funnel
  • Filter paper
  • Vacuum pump
  • 5.5 × 5–cm, 4.0 × 12–cm, and 1.5 × 25–cm silica gel columns
  • NMR (Avance III 400, Bruker)
  • Ground‐glass stoppers
  • Precision pH test paper
  • 10‐mL constant pressure funnel
  • Rubber stopper or Parafilm
CAUTION: Most of the organic solvents are flammable.

Basic Protocol 2: Synthesis, Purification, and Characterization of Caged RNA Oligoribonucleotides

  Materials
  • Photolinker (PL) phosphoramidite (see protocol 1)
  • Vitamin E phosphoroamidites (see protocol 1)
  • Commercially available nucleoside 3′‐phosphoramidites (WuhuHuaren):
    • Adenosine, N‐benzoyl‐5′‐O‐[bis(4‐methoxyphenyl)phenylmethyl]‐2′‐O‐[(1,1‐dimethylethyl)dimethylsilyl], 3′‐[2‐cyanoethyl N,N‐bis(1‐methylethyl)phosphoramidite]
    • Cytidine, N‐benzoyl‐5′‐O‐[bis(4‐methoxyphenyl)phenylmethyl]‐2′‐O‐[(1,1‐dimethylethyl)dimethylsilyl], 3′‐[2‐cyanoethyl N,N‐bis(1‐methylethyl)phosphoramidite]
    • Guanosine, N‐2‐methyl‐1‐oxopropyl‐5′‐O‐[bis(4‐methoxyphenyl)phenylmethyl]2′‐O‐1,1‐dimethylethyl)dimethylsilyl] 3′‐[2‐cyanoethyl N,N‐bis(1‐methylethyl)phosphoramidite]
    • Uridine, 5′‐O‐bis(4‐methoxyphenyl)phenylmethyl]‐2′‐O‐[(1,1‐dimethylethyl)dimethylsilyl] 3′‐[2‐cyanoethyl N,N‐bis(1‐methylethyl)phosphoramidite]
    • Thymidine, 5′‐O‐[bis(4‐methoxyphenyl)phenylmethyl] 3′‐[2‐cyanoethyl N,N‐bis(1‐methylethyl)phosphoramidite]
  • Acetonitrile (CH 3CN, anhydrous 10 ppm, Shanghai Pudi Biotechnology)
  • Nitrogen source
  • 3′‐dT CPG (Hai Phoenix)
  • Acetonitrile (CH 3CN, HPLC grade)
  • 33% ammonium hydroxide solution
  • Dimethyl sulfoxide (DMSO, Aldrich)
  • (C 2H 5) 3 N⋅3HF (TEA⋅3HF, 98% Aldrich)
  • Sodium acetate (3 M aqueous solution)
  • n‐Butyl alcohol
  • Diethyl pyrocarbonate (DEPC, Aldrich)
  • Pure water (Quchenshi)
  • Ammonium bicarbonate (2 M aqueous solution, add CO 2 to 2 M TEA aqueous solution and adjust pH to 8.0)
  • 1% triethyl amine
  • PBS
  • 40% acrylamide/bisacrylamide (19:1) (Dingguochangshen)
  • 1× TBE running buffer
  • Urea (electrophoresis‐grade, Vetec)
  • Ammonium persulfate (APS, Sinopharm Chemical Reagent)
  • N,N,N′,N′‐Tetramethylethylenediamine (TEMED, >99%, HARVEYBIO)
  • 6× RNA loading buffer (Dingguochangshen)
  • Reference markers
  • SYBR gold (Invitrogen)
  • DEPC water: add 1 mL DEPC to 1 L water, stir overnight, and sterilize by autoclaving for 15 min
  • Automated oligonucleotide synthesizer (Applied Biosystems 394)
  • 200‐μL and 1.5‐mL microcentrifuge tubes
  • Parafilm
  • Oscillator
  • SpeedVac evaporator (Thermo)
  • Vacuum pump and water aspirator
  • Vortex mixer
  • 35°C shaker
  • Refrigerated microcentrifuge
  • HPLC system (Waters, Alliance e2695)
  • Semi‐preparative column with reverse‐phase adsorption of mixed type anion exchange solid‐phase extraction (Waters XBridgeTM OST C18)
  • UV/vis spectrophotometer (NanoDrop ND‐1000, Thermo)
  • Conical vials
  • Mass spectrometer
  • Gel plates
  • 0.75‐mm spacers
  • 0.75‐mm‐thick combs
  • Syringe filter (PES, 13 mm, 0.22 µm)
  • Gel electrophoresis equipment (Bio‐Rad Mini‐PROTEAN Tetra system)
  • 96‐well plate (Costar)
  • LED lamp (Lamplic,cat. no. UVDRV‐8‐A‐0150‐30)
  • Chemiluminescence gel imaging system

Basic Protocol 3: Photochemical Regulation of Gene Expression Using Caged siRNAs for Reporter Genes (GFP/RFP and Firefly/Renilla Luciferase)

  Materials
  • LB solid powder (Tiandz)
  • Sodium hydroxide (NaOH, analytical purity)
  • Agar powder (Solarbio)
  • Kanamycin (biotechnology grade, MP)
  • Ampicillin (biotechnology grade, Amresco)
  • BL21 (DE3) chemically competent cells (Dingguo)
  • GFP plasmid (pEGFP‐N1, Sangon Biotech)
  • RFP plasmid (pDsRed‐N1, Sangon Biotech)
  • Firefly plasmid (pGL‐3, Sangon Biotech)
  • Renilla plasmid (pRL‐TK, Sangon Biotech)
  • Glycerol (Macklin)
  • PureYield Plasmid Maxiprep System (Promega)
  • Nuclease‐free water
  • Human embryonic kidney cells (HEK293 cells)
  • Dulbecco's modified Eagle's medium (DMEM, M&C)
  • Defined fetal bovine serum (FBS)
  • Penicillin‐streptomycin (100 μg/mL, M&C)
  • Phosphate buffer saline (PBS, M&C)
  • Trypsin‐EDTA (M&C)
  • Lipofectamine2000 (Invitrogen)
  • Opti‐MEM (Life)
  • Dual‐Luciferase Reporter Assay System (Promega)
  • 250‐ and 500‐mL conical flasks
  • Hotplate
  • Oscillator
  • Autoclave
  • 90‐mm culture dishes
  • 42°C water bath
  • Bacteriological incubator shaker (ZHICHENG, cat. no. ZWY‐211 C)
  • Inoculating loops
  • 15‐, 50‐, and 500‐mL centrifuge tubes (Axygen)
  • UV/vis spectrophotometer (NanoDrop ND‐1000, Thermo)
  • Centrifuge (Eppendorf)
  • Vacuum manifold
  • Vacuum
  • 75‐cm2 cell culture flask (Trueline)
  • 37°C, 5% CO 2 humidified incubator (Thermo)
  • Cell counting chamber
  • 6‐ and 24‐well cell culture plates (Costar)
  • LED‐UV (Berthold Technologies)
  • 96‐well luminescence test plate
  • High‐sensitivity microplate luminometer (Centro XS3 LB 900, Berthold Technologies)
  • Automatic inverted fluorescence microscope (Olymups model no. IX83)
  • 400‐mesh screen
  • Flow cytometry (BD FACSAriaII)

Basic Protocol 4: Intracellular Distribution and Cellular Uptake Pathways

  Materials
  • Human embryonic kidney cells (HEK293 cells)
  • Dulbecco's modified Eagle's medium (DMEM, M&C)
  • Lipofectamine2000 (Invitrogen)
  • Opti‐MEM (Life)
  • Firefly and renilla plasmids (see protocol 3)
  • Cy3‐labeled sense RNA of luciferase siRNA (Zixibio)
  • Phosphate buffer saline (PBS, M&C)
  • Hoechst33342 (Sigma)
  • Chlorpromazine HCl (HARVEYBIO)
  • Dimethylsulfoxide (DMSO, cell culture grade)
  • Amiloride hydrochloride (HARVEYBIO)
  • Methyl‐β‐cycloetrin (HARVEYBIO)
  • Genistein (HARVEYBIO)
  • Trypsin‐EDTA (M&C)
  • Laser confocal culture dishes (35‐mm)
  • 37°C, 5% CO 2 incubator (Thermo)
  • 1.5‐mL microcentrifuge tubes (Axygen)
  • Vortex mixer
  • Laser scanning confocal microscopy (Nikon,A1R)
  • 6‐well plates
  • 37°C water bath
  • Centrifuge (Eppendorf)
  • 400‐mesh screen
  • Cell counting chamber
  • Flow cytometry (BD FACSAriaII)
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Figures

Videos

Literature Cited

Literature Cited
  Elbashir, S.M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., and Tuschl, T. 2001. Duplexes of 21‐nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411:494‐498. doi: 10.1038/35078107.
  Govan, J.M., Young, D.D., Lusic, H., Liu, Q., Lively, M.O., and Deiters, A. 2013. Optochemical control of RNA interference in mammalian cells. Nucleic Acids Res. 41:10518‐10528. doi: 10.1093/nar/gkt806.
  Jain, P.K., Shah, S., and Friedman, S.H. 2011. Patterning of gene expression using new photolabile groups applied to light activated RNAi. J. Am. Chem. Soc. 133:440‐446. doi: 10.1021/ja107226e.
  Ji, Y., Yang, J., Wu, L., Yu, L., and Tang, X. 2016. Photochemical regulation of gene expression using caged siRNAs with single terminal vitamin E modification. Angew. Chem. Int. Ed. 55:2152‐2156. doi: 10.1002/anie.201510921.
  Mikat, V. and Heckel, A. 2007. Light‐dependent RNA interference with nucleobase‐caged siRNAs. RNA 13:2341‐2347. doi: 10.1261/rna.753407.
  Nguyen, Q.N., Chavli, R.V., Marques, J.T., Conrad Ii, P.G., Wang, D., He, W., Belisle, B.E., Zhang, A., Pastor, L.M., Witney, F.R., Morris, M., Heitz, F., Divita, G., Williams, B.R.G., and McMaster, G.K. 2006. Light controllable siRNAs regulate gene suppression and phenotypes in cells. Biochim. Biophys. Acta– Biomem. 1758:394‐403. doi: 10.1016/j.bbamem.2006.01.003.
  Shah, S., Jain, P.K., Kala, A., Karunakaran, D., and Friedman, S.H. 2009. Light‐activated RNA interference using double‐stranded siRNA precursors modified using a remarkable regiospecificity of diazo‐based photolabile groups. Nucleic Acids Res. 37:4508‐4517. doi: 10.1093/nar/gkp415.
  Wu, L., Pei, F., Zhang, J., Wu, J., Feng, M., Wang, Y., Jin, H., Zhang, L., and Tang, X. 2014. Synthesis of site‐specifically phosphate‐caged siRNAs and evaluation of their RNAi activity and stability. Chem. Eur. J. 20:12114‐12122. 10.1002/chem.201403430
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