Synthesis of Site‐Specifically Phosphate‐Caged siRNAs

Li Wu1, Jie Wang2, Xinjing Tang2

1 School of Chemistry and Chemical Engineering, University of Chinese Academy of Sciences, Beijing, 2 State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 6.12
DOI:  10.1002/0471142700.nc0612s61
Online Posting Date:  June, 2015
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Abstract

Photolabile small interfering RNA (siRNA) oligonucleotide duplexes are becoming a powerful tool for photoregulation of gene expression through an RNA interference (RNAi) mechanism. Terminal or statistical labeling of siRNAs has been previously achieved. Recently, we have shown a new strategy for site‐specific incorporation of a photolabile group (1‐(2‐nitrophenyl)ethyl [NPE]) at any phosphate position of siRNA strands for photomodulation of their gene‐silencing activity. In this unit, we first describe in detail the syntheses of four new NPE protected nucleoside phosphoramidites (dA0, dG0, dC0, dT0) and 2‐cyanoethyl‐1‐(2‐nitrophenyl)ethyl‐N,N′‐diisopropylphosphoramidite (N0). They are then site specifically incorporated into any position of RNA oligonucleotides according to standard phosphoramidite chemistry. Phosphate‐caged siRNA duplexes are then prepared by hybridization of single‐stranded RNAs containing the 1‐(2‐nitrophenyl)ethyl caging moiety on the phosphate group with their complementary RNA. © 2015 by John Wiley & Sons, Inc.

Keywords: uncaging; photoregulation; site‐specific; phosphate‐caged siRNA; RNAi

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Photolabile Nucleotide Phosphoramidites
  • Basic Protocol 2: Synthesis of 2‐Cyanoethyl‐1‐(2‐Nitrophenyl)Ethyl‐N,N′‐Diisopropylphosphoramidite
  • Basic Protocol 3: Synthesis of Phosphate‐Caged RNA Oligonucleotides
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of Photolabile Nucleotide Phosphoramidites

  Materials
  • Commercially available protected nucleosides:
    • 5′‐O‐(4,4′‐dimethoxytrityl)‐N6‐benzoyl adenosine (Wuhu Huaren Scientific)
    • 5′‐O‐(4,4′‐dimethoxytrityl) thymidine(Wuhu Huaren Scientific)
    • 5′‐O‐(4,4′‐dimethoxytrityl)‐N4‐benzoyl cytidine (Wuhu Huaren Scientific)
    • 5′‐O‐(4,4′‐dimethoxytrityl)‐N2‐isobutyryl guanosine (Wuhu Huaren Scientific)
  • 1‐(2‐Nitrophenyl)ethanol (NPE)
  • Bis(diisopropylamino)chlorophosphine (>97%, Sigma‐Aldrich)
  • Calcium hydride (CaH 2)
  • Tetrazole
  • Diisopropylethylamine (DIPEA, analytical grade; freshly distilled and stored over CaH 2)
  • Dichloromethane (DCM, analytical grade; freshly distilled and stored over CaH 2)
  • Tetrahydrofuran (THF, analytical grade; freshly distilled and stored over CaH 2)
  • Triethylamine (TEA, analytical grade; freshly distilled and stored over CaH 2)
  • Ethyl acetate (EA, analytical grade; freshly distilled and stored over CaH 2)
  • Petroleum ether (PE, analytical grade; freshly distilled and stored over CaH 2)
  • Anhydrous acetonitrile (cat. no. 364310010, Acros Organics)
  • Chloroform‐d (CDCl 3)
  • Silica gel (Merck, type 9385, 230 to 400 mesh)
  • Thin‐layer chromatography (TLC) plates (silica gel 60 F, Merck)
  • Sand (for filtration)
  • Nitrogen (N 2; or argon) gas, dry
  • Rotary evaporator
  • Vacuum pump
  • Magnetic stir bars and plate
  • Syringes and needles
  • 2 × 20–cm silica gel column
  • 25‐, 50‐ and 100‐mL round‐bottom flasks
  • UV hand lamp
  • Sand core funnel
  • Suction flask
  • Syringe filter
  • Nuclear magnetic resonance spectrometer (Avance III 400, Bruker)

Basic Protocol 2: Synthesis of 2‐Cyanoethyl‐1‐(2‐Nitrophenyl)Ethyl‐N,N′‐Diisopropylphosphoramidite

  Materials
  • 1‐(2‐Nitrophenyl)ethanol (NPE)
  • Bis(diisopropylamino)(2‐cyanoethoxy)phosphine
  • Calcium hydride (CaH 2)
  • Tetrazole
  • Tetrahydrofuran (THF, analytical grade; freshly distilled and stored over CaH 2)
  • Dichloromethane (DCM, analytical grade, freshly distilled over CaH 2; Beijing TongGuang)
  • Triethylamine (TEA, analytical grade, freshly distilled over CaH 2; Beijing TongGuang),
  • Ethyl acetate (EA, analytical grade, freshly distilled over CaH 2; Beijing TongGuang)
  • Petroleum ether (PE, analytical grade, freshly distilled over CaH 2; Beijing TongGuang)
  • Chloroform‐d (CDCl 3)
  • Silica gel (230 to 400 mesh, Merck Grade 9385))
  • Thin‐layer chromatography (TLC) plates (silica gel 60 F, Merck)
  • Nitrogen (N 2; or argon) gas, dry
  • Rotary evaporator
  • Vacuum pump and water aspirator
  • Magnetic stir bars and plate
  • Pipetman (optional)
  • 2 × 20–cm silica gel column
  • 25‐, 50‐ and 100‐mL round‐bottom flasks
  • UV hand lamp
  • Nuclear magnetic resonance spectrometer (Avance III 400, Bruker)

Basic Protocol 3: Synthesis of Phosphate‐Caged RNA Oligonucleotides

  Materials
  • Commercially available nucleoside 3′‐phosphoramidites (Wuhu Huaren Scientific):
    • 5′‐O‐(4,4′‐dimethoxytrityl)‐2′‐O‐(tert‐butyldimethylsilyl)‐N6‐benzoyladenosine‐3′‐(2‐cyanoethyl‐N,N‐diisopropyl) phosphoramidite
    • 5′‐O‐(4,4′‐dimethoxytrityl)‐2′‐O‐(tert‐butyldimethylsilyl)‐thymidine‐3′‐(2‐cyanoethyl‐N,N‐diisopropyl) phosphoramidite
    • 5′‐O‐(4,4′‐dimethoxytrityl)‐2′‐O‐(tert‐butyldimethylsilyl)‐N4‐benzoylcytidine‐3′‐(2‐cyanoethyl‐N,N‐diisopropyl) phosphoramidite
    • 5′‐O‐(4,4′‐dimethoxytrityl)‐2′‐O‐(tert‐butyldimethylsilyl)‐N2‐isobutyrylguanosine‐3′‐(2‐cyanoethyl‐N,N‐diisopropyl) phosphoramidite
    • 5′‐O‐(4,4′‐dimethoxytrityl)‐2′‐fluoro‐N6‐benzoyladenosine‐3′‐(2‐cyanoethyl‐N,N‐diisopropyl) phosphoramidite
    • 5′‐O‐(4,4′‐dimethoxytrityl)‐2′‐fluoro‐thymidine‐3′‐(2‐cyanoethyl‐N,N‐diisopropyl) phosphoramidite
    • 5′‐O‐(4,4′‐dimethoxytrityl)‐2′‐fluoro‐N4‐benzoylcytidine‐3′‐(2‐cyanoethyl‐N,N‐diisopropyl) phosphoramidite
    • 5′‐O‐(4,4′‐dimethoxytrityl)‐2′‐fluoro‐N2‐isobutyrylguanosine‐3′‐(2‐cyanoethyl‐N,N‐diisopropyl) phosphoramidite
  • Modified nucleoside 3′‐phosphoramidites:
    • 5′‐O‐(4,4′‐dimethoxytrityl)‐N6‐benzoyladenosine‐3′‐{O‐[1‐(2‐nitrophenyl)ethyl]‐N,N′‐diisopropyl} phosphoramidite (dA0; see protocol 1)
    • 5′‐O‐(4,4′‐dimethoxytrityl)‐thymidine‐3′‐{O‐[1‐(2‐nitrophenyl)‐ethyl]‐N,N′‐diisopropyl} phosphoramidite (dT0; see Basic Protocol 1)  
    • 5′‐O‐(4,4′‐dimethoxytrityl)‐N4‐benzoylcytidine‐3′‐{O‐[1‐(2‐nitrophenyl)ethyl]‐N,N′‐diisopropyl} phosphoramidite (dC0; see protocol 1)
    • 5′‐O‐(4,4′‐dimethoxytrityl)‐N2‐isobutyrylguanosine‐3′‐{O‐[1‐(2‐nitrophenyl)ethyl]‐N,N′‐diisopropyl} phosphoramidite (dG0; see protocol 1)
    • Phosphoramidous acid, N,N‐bis(1‐methylethyl)‐2‐({2‐[bis(4‐methoxyphenyl)(phenyl)methoxy]ethyl}sulfonyl)ethyl 2‐cyanoethyl ester
  • Acetonitrile (CH 3CN, HPLC grade)
  • Acetonitrile (CH 3CN, anhydrous [10 ppm, the amount of water present]; SuZhou Yafan)
  • Dimethyl sulfoxide (DMSO; Sigma‐Aldrich).
  • Triethylamine trihydrofluoride (TEA·3HF, 98%; Sigma‐Aldrich)
  • Triethylammonium bicarbonate (TEAB) buffer (2 M aqueous solution: add CO 2 to 2 M triethylamine aqueous solution and adjust pH to 8.0; dilute 2 M TEAB solution with DEPC treated water to a final concentration of 0.05 M TEAB; 0.05 M TEAB buffer used for purification by HPLC)
  • Sodium acetate (NaOAc, 3 M aqueous solution)
  • n‐Butyl alcohol
  • Diethyl pyrocarbonate (DEPC, Sigma‐Aldrich)
  • Diethyl pyrocarbonate (DEPC) treated water (mix 1:1000 DEPC/HPLC‐grade water; sterilize at high temperature)
  • Methylamine solution, 33 wt.% in ethanol (Sigma‐Aldrich)
  • Gel loading buffer
  • Formamide (Sigma‐Aldrich)
  • EDTA (Sigma‐Aldrich)
  • Urea (Sigma‐Aldrich)
  • Bromophenol blue (Sigma‐Aldrich)
  • Xylene cyanol (Sigma‐Aldrich)
  • 1× TBE running buffer
  • 6× RNA loading buffer
  • SYBR gold nucleic acid gel stain (Life Technologies)
  • Vacuum pump and water aspirator
  • Oscillator
  • Vortex finder
  • Automated oligonucleotide synthesizer (Applied Biosystems Model 394 or equivalent)
  • Parafilm
  • −80°C freezer
  • 10‐mL disposable syringes
  • SepPak cartridges
  • Speedvac evaporator (Thermo Scientific)
  • Microcentrifuge or EP tubes (Eppendorf)
  • Semi‐preparative column with reversed‐phase adsorption for mixed‐type anion exchange solid‐phase extraction (Waters XBridge OST C18, Waters)
  • HPLC (Alliance HPLC system with e2695 separations module, Waters)
  • UV/VIS spectrophotometer (DU 800 series, Beckman Coulter)
  • UV/VIS spectrophotometer (NanoDrop ND‐1000, Thermo Scientific)
  • Mass spectrometer with electrospray ionization (ESI; Waters Xevo G2 Q‐Tof)
  • Chemiluminescence gel imaging system (ChemiDoc XRS)
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Figures

Videos

Literature Cited

Literature Cited
  Beaucage, S.L. and Caruthers, M.H. 2001. Synthetic strategies and parameters involved in the synthesis of oligodeoxyribonucleotides according to the phosphoramidite method. Curr. Protoc. Nucl. Acid Chem. 00:3.3.1‐3.3.20. doi:10.1002/0471142700.nc0303s00.
  Blidner, R.A., Svoboda, K.R., Hammer, R.P., and Monroe, T.W. 2008. Photoinduced RNA interference using DMNPE‐caged 2′‐deoxy‐2′‐fluoro substituted nucleic acids in vitro and in vivo. Mol. Biosyst. 4:431‐440.
  Chen, M., Zhang, L., Zhang, H.‐Y., Xiong, X., Wang, B., Du, Q., Lu, B., Wahlestedt, C., and Liang, Z. 2005. A universal plasmid library encoding all permutations of small interfering RNA. Proc. Natl. Acad. Sci. U.S.A. 102:2356‐2361.
  Chiu, Y.L. and Rana, T.M. 2002. RNAi in human cells: Basic structural and functional features of small interfering RNA. Mol. Cell 10:549‐561.
  Chu, Y.L. and Rana, T.M. 2003. siRNA function in RNAi: A chemical modification analysis. RNA 9:1034‐1048.
  Connelly, C.M., Uprety, R., Hemphill, J., and Deiters, A. 2012. Spatiotemporal control of microRNA function using light‐activated antagomirs. Mol. Biosyst. 8:2987‐2993.
  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.
  Mikat, V. and Heckel, A. 2007. Light‐dependent RNA interference with nucleobase‐caged siRNAs. RNA 13:2341‐2347.
  Rana, T.M. 2007. Illuminating the silence: Understanding the structure and function of small RNAs. Nat. Rev. Mol. Cell Biol. 8:23‐36.
  Shah, S., Rangarajan, S., and Friedman, S.H. 2005. Light‐activated RNA interference. Angew. Chem. Int. Ed. 44:1328‐1332.
  Su, M., Wang, J., and Tang, X. 2012. Photocaging strategy for functionalisation of oligonucleotides and its applications for oligonucleotide labelling and cyclisation. Chem. Eur. J. 18:9628‐9637.
  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.
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