Preparation of Short Interfering RNA Containing the Modified Nucleosides 2‐Thiouridine, Pseudouridine, or Dihydrouridine

Barbara Nawrot1, Elzbieta Sochacka2

1 Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Lodz, Poland, 2 Institute of Organic Chemistry, Technical University of Lodz, Lodz, Poland
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 16.2
DOI:  10.1002/0471142700.nc1602s37
Online Posting Date:  June, 2009
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


Modified uridine derivatives such as 2‐thiouridine (s2U), pseudouridine (Ψ), and dihydrouridine (D) are naturally existing nucleoside units identified in tRNA molecules. Recently, we have shown that such base‐modified units introduced into functionally important sites of siRNA modulate thermodynamic stability of the duplex and its gene silencing activity. In this unit, we describe chemical synthesis of 3′‐phosphoramidite derivatives of s2U and D units (the 3′‐phosphoramidite derivative of Ψ is commercially available), and their use for the synthesis of RNA oligonucleotides according to the routine phosphoramidite protocol. The only exception concerns the oxidation step with I2/pyridine/water which, if applied towards oligonucleotides containing s2U units, would lead to the loss of sulfur. Therefore, to avoid this side reaction, tert‐butyl hydroperoxide is used as an oxidizing reagent. After the oligonucleotide chain assembly is completed, the resulting oligomer is deprotected under mild basic conditions (MeNH2/EtOH/DMSO) to avoid dihydrouracil ring opening, which is a reported side‐reaction during the routine synthesis of dihydrouridine‐containing RNA. Oligonucleotides modified with s2U, D, or Ψ units are useful models for structure‐function studies. Here, the procedure for preparation of siRNA duplexes is described. Curr. Protoc. Nucleic Acid Chem. 37:16.2.1‐16.2.16. © 2009 by John Wiley & Sons, Inc.

Keywords: 2‐thiouridine; dihydrouridine; pseudouridine; RNA synthesis; siRNA; small interfering RNA; phosphoramidite synthesis

PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesis of Dihydrouridine and 2‐Thiouridine 3′‐Phosphoramidites
  • Basic Protocol 2: Synthesis of Oligonucleotides Containing Modified 2‐Thiouridine, Pseudouridine, or Dihydrouridine, and Preparation of Short Interfering RNAs
  • Commentary
  • Literature Cited
  • Figures
  • Tables
PDF or HTML at Wiley Online Library


Basic Protocol 1: Synthesis of Dihydrouridine and 2‐Thiouridine 3′‐Phosphoramidites

  • Modified nucleosides:
    • 5,6‐Dihydrouridine (International Laboratory Limited or Chemcube, Niederkassel, Germany)
    • 2‐Thiouridine (Berry and Associates, International Laboratory Limited, or Chemcube, Niederkassel, Germany)
  • Pyridine (anhydrous, analytical grade; Aldrich), freshly distilled over CaH 2
  • 4,4′‐Dimethoxytrityl chloride (DMTr‐Cl; Aldrich)
  • Silica gel (Merck, type 9385, 230 to 400 mesh)
  • Dichloromethane
  • Methanol
  • 5% aqueous sodium bicarbonate (NaHCO 3)
  • Sodium sulfate (anhydrous)
  • Toluene
  • Triethylamine (TEA, anhydrous, HPLC grade; Aldrich)
  • Tetrahydrofurane (anhydrous)
  • Silver nitrate (Aldrich)
  • tert‐ Butyldimethylsilyl chloride (TBDMS‐Cl; Aldrich)
  • Acetone
  • Hexane
  • Ethyl acetate
  • Imidazole (Aldrich)
  • Benzene
  • Argon (or nitrogen) gas, dry
  • Desiccant silica gel (Aldrich)
  • N,N‐Diisopropylethylamine (DIPEA, anhydrous; Aldrich)
  • 2‐Cyanoethyl N,N‐diisopropyl chlorophosphoramidite (Aldrich)
  • Acetic anhydride (Ac 2O)
  • N,N‐Dimethylaminopyridine (DMAP; 99%; Aldrich)
  • Ethyl ether
  • Petroleum ether
  • Rotary evaporator
  • Vacuum pump and water aspirator
  • Magnetic stir bars and plate
  • Thin‐layer chromatography (TLC) plates (Silica gel 60F, Merck)
  • 200‐mL and 100‐mL separatory funnels
  • 5.5 × 5–cm, 4.0 × 12–cm, and 1.5 × 25–cm silica gel columns
  • 25‐, 50‐ and 100‐mL round–bottom flasks
  • Rubber septa
  • Luer‐lock needles with syringes
  • 1‐mL, 500‐µL and 200‐µL syringes with needles
  • Glass capillaries
  • Rubber cork or Parafilm
  • Vacuum desiccators
  • Additional reagents and equipment for TLC ( appendix 3D)

Basic Protocol 2: Synthesis of Oligonucleotides Containing Modified 2‐Thiouridine, Pseudouridine, or Dihydrouridine, and Preparation of Short Interfering RNAs

  • Commercial nucleoside 3′‐phosphoramidites:
    • 5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐O‐(tert‐butyldimethylsilyl)‐3′‐O‐[(2‐cyanoethoxy)‐(N,N‐diisopropylamino)]phosphinyl uridine and N‐protected cytidine, adenosine, and guanosine (Glen Research)
    • 5′‐O‐(4,4′‐Dimethoxytrityl)‐3′‐O‐[(2‐cyanoethoxy)‐(N,N‐diisopropylamino)] phosphinyl thymidine (Glen Research)
  • Modified nucleoside 3′‐phosphoramidites:
    • 5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐O‐(tert‐butyldimethylsilyl)‐3′‐O‐[(2‐cyanoethoxy)‐(N,N‐diisopropylamino)]phosphinyl 5,6‐dihydrouridine (S.4a prepared according to protocol 1)
    • 5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐O‐(tert‐butyldimethylsilyl)‐3′‐O‐[(2‐cyanoethoxy)‐(N,N‐diisopropylamino)]phosphinyl 2‐thiouridine (S.4b prepared according to protocol 1)
    • 5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐O‐(tert‐butyldimethylsilyl)‐3′‐O‐[(2‐cyanoethoxy)‐(N,N‐diisopropylamino)]phosphinyl pseudouridine (Glen Research)
  • Acetonitrile (CH 3CN), anhydrous (Fluka)
  • Tert‐butyl hydroperoxide (tBuOOH) ∼5.5 M solution in decane (over molecular sieve 4Å; Aldrich)
  • Methylamine, 33% ethanolic solution (Aldrich)
  • Dimethyl sulfoxide (DMSO; Aldrich)
  • (C 2H 5) 3N × 3HF 98% (TEA × 3HF; Aldrich, cat. no. 344648)
  • Ammonium bicarbonate
  • Sodium acetate (NaOAc), anhydrous (Sigma)
  • Acetate buffer (mixture of 50 mM NaOAc and 50 mM NaCl)
  • Trifluoroacetic acid, TFA (Fluka)
  • NaCl
  • Diethyl pyrocarbonate (DEPC; Aldrich)
  • Milli‐Q water
  • Automated oligonucleotide synthesizer (Applied Biosystems 394 or equivalent)
  • 4‐mL screw‐top vials
  • 65°C heating block
  • −20°C freezer
  • 10‐mL disposable syringes
  • SepPak cartridges (Waters)
  • Speedvac evaporator (Savant)
  • UV/VIS spectrophotometer (NanoDrop ND‐1000)
  • Autoclave (e.g., Varioklav Model 75T or 135T, H+P Labortechnik AG, Germany)
  • Microcentrifuge tubes (e.g., Eppendorf)
  • 95°C water bath
  • Additional reagents and equipment for automated oligoribonucleotide synthesis (see appendix 3C), purifying oligonucleotides using RP‐HPLC (unit 10.5) or PAGE (unit 10.4)
PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
   Agris, P.F. 1996. Modified nucleosides in RNA structure and function. In Encyclopedia of NMR (D. M. Grant and R.K. Harris, eds.; Chan, S., section ed.) pp. 4151‐4158. John Wiley & Sons, UK.
   Agris, P.F., Sierzputowska‐Gracz, H., Smith, W., Malkiewicz, A., Sochacka, E., and Nawrot, B. 1992. Thiolation of Uridine Carbon‐2 Restricts the Motional Dynamics of the Transfer RNA Wobble Position Nucleoside. J. Am. Chem. Soc. 114:2652‐2656.
   Agris, P.F., Malkiewicz, A., Kraszewski, A., Everett, K., Nawrot, B., Sochacka, E., Jankowska, J., and Guenther, R. 1995. Site‐specific introduction of modified purine and pyrimidine ribonucleosides into RNA by automated phosphoramidite chemistry. Biochimie 77:125‐134.
   Beaucage, S.L. and Caruthers, M.H. 1981. Deoxynucleoside phosphoramidities—a new class of key intermediates for deoxypolynucleotide synthesis. Tetrahedron Lett. 22:1859‐1862.
   Bittker, J.A., Philips, K.J., and Liu, D.R. 2002. Recent advances in the in vitro evolution of nucleic acids. Curr. Opin. Chem. Biol. 6:367‐374.
   Brown, T. and Brown, D.J.S. 1991. Modern machine‐aided methods of oligodeoxyribonucleotide synthesis. In Oligonucleotides and Analogues. A Practical Approach (F. Eckstein, ed.) pp. 1‐25. Oxford University Press, Oxford.
   Chaix, C., Duplaa, A.M., Molko, D., and Teoule, R. 1989. Solid phase synthesis of the 5′‐half of the initiator t‐RNA from B. subtilis. Nucleic Acids Res. 17:7381‐7393.
   Chiu, Y.L. and Rana, T.M. 2003. siRNA function in RNAi: A chemical modification analysis. RNA 9:1034‐1048.
   Cummins, L.L., Owen, S.R., Risen, L.M., Lesnik, E.A., Freier, S.M., McGee, D., Guinosso, C.J., and Cook, P.D. 1995. Characterization of fully 2′‐modified oligoribonucleotide hetero‐ and homoduplex hybridization and nuclease sensitivity. Nucleic Acids Res. 23:2019‐2024.
   Dalluge, J.J., Hashizume, T., Sopchik, A.E., McCloskey, J.A., and Davis, D.R. 1996. Conformational flexibility in RNA: The role of dihydrouridine. Nucleic Acids Res. 24:1073‐1079.
   Davis, D. 1998. Biophysical and conformational properties of modified nucleosides in RNA (magnetic resonance studies). In Modification and Editing of RNA (H. Grosjean and B. Benne, eds.) pp. 85‐102. ASM Press, Washingon D.C.
   Davis, D.R. and Bajji, A.C. 2005. Introduction of hypermodified nucleotides in RNA. Methods Mol. Biol. 288:187‐204.
   Davis, D.R., Veltri, C.A., and Nielsen, L. 1998. An RNA model system for investigation of pseudouridine stabilization of the codon‐anticodon interaction in tRNALys, tRNAHis and tRNATyr. J. Biomol. Struct. Dyn. 15:1121‐1132.
   Durant, P.C., Bajji, A.C., Sundaram, M., Kumar, R.K., and Davis, D.R. 2005. Structural effects of hypermodified nucleosides in the Escherichia coli and human tRNALys anticodon loop: The effect of nucleosides s2U, mcm5U, mcm5s2U, mnm5s2U, t6A, and ms2t6A. Biochemistry 44:8078‐8089.
   Earnshaw, D.J. and Gait, M.J. 1998. Modified oligoribonucleotides as site‐specific probes of RNA structure and function. Biopolymers 48:39‐55.
   Elbashir, S.M., Martinez, J., Patkaniowska, A., Lendecel, W., and Tuschl, T. 2001a. Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate. EMBO J. 20:6877‐6888.
   Elbashir, S.M., Lendeckel, W., and Tuschl, T. 2001b. RNA interference is mediated by 21‐ and 22‐nucleotide RNAs. Genes Dev. 15:188‐200.
   Elbashir, S.M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., and Tuschl, T. 2001c. Duplexes of 21‐nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411:494‐498.
   Elbashir, S.M., Martinez, J., Patkaniowska, A., Lendecel, W., and Tuschl, T. 2001d. Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate. EMBO J. 20:6877‐6888.
   Fire, A., Xu, S.Q., Montgomery, M.K., Kostas, S.A., Driver, S.E., and Mello, C.C. 1998. Potent and specific genetic interference by double‐stranded RNA in Caenorhabitis elegans. Nature 391:806‐813.
   Fougerolles, A., Manoharan, M., Meyers, R., and Vornlocher, H‐P. 2005. RNA interference in vivo: Towards synthetic small inhibitory RNA‐based therapeutics. Methods Enzymol. 392:278‐296.
   Gasparutto, D., Livache, T., Bazin, H., Duplaa, A.M., Guy, A., Khorlin, A., Molko, D., Roget, A., and Teoule, R. 1992. Chemical synthesis of a biologically active natural tRNA with its minor bases. Nucleic Acids Res. 20:5159‐5156.
   Grasby, J.A. and Gait, M.J. 1994. Synthetic oligoribonucleotides carrying site‐specific modifications for RNA structure‐function analysis. Biochimie 76:1223‐1234.
   Guenther, R.H., Bakal, R.S., Forrest, B., Chen, Y., Sengupta, R., Nawrot, B., Sochacka, E., Jankowska, J., Kraszewski, A., Malkiewicz, A., and Agris, P.F. 1994. Aminoacyl‐tRNA synthetase and U54 methyltransferase recognize conformations of the yeast tRNA(Phe) anticodon and T stem/loop domain. Biochimie 76:1143‐1151.
   Hakimelahi, G.H., Proba, Z.A., and Ogilvie, K.K. 1982. New catalysts and procedures for the dimethoxytritylation and selective silylation of ribonucleosides. Can. J. Chem. 60:1106‐1113.
   Hanze, A.R. 1967. Nucleic acids. IV. The catalytic reduction of pyrimidine nucleosides (human liver deaminase inhibitors). J. Am. Chem. Soc. 89:6720‐6725.
   Karikó, K., Buckstein, M., Ni, H., and Weissman, D. 2005. Suppression of RNA recognition by Toll‐like receptors: The impact of nucleoside modification and the evolutionary origin of RNA. Immunity 23:165‐175.
   Ketting, R.F., Fischer, S.E., Bernstein, E., Sijen, T., Hannon, G.J., and Plasterk, R.H. 2001. Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C. elegans. Genes Dev. 15:2654‐2659.
   Khvorova, A., Reynolds, A., and Jayasena, S.D. 2003. Functional siRNAs and miRNAs exhibit strand bias. Cell 115:209‐216.
   Kumar, R.K. and Davis, D.R. 1995. Synthesis of oligoribonucleotides containing 2‐thiouridine: Incorporation of 2‐thiouridine phosphoramidite without base protection. J. Org. Chem. 60:7726‐7727.
   Kumar, R.K. and Davis, D.R. 1997. Synthesis and studies on the effect of 2‐thiouridine and 4‐thiouridine on sugar conformation and RNA duplex stability. Nucleic Acids Res. 25:1272‐1280.
   Leake, D., Reynolds, A., Khvorova, A., Marshall, W., and Scaringe, S. 2004. Stabilized polynucleotides for use in RNA interference. US Patent 2004/0198640 A1.
   Lescrinier, E., Nauwelaerts, K., Zanier, K., Poesen, K., Sattier, M., and Herdewijn, P. 2006. The naturally occurring N6‐threonyl adenine in anticodon loop of Schizosaccharomyces pombe tRNAi causes of a unique U‐turn motif. Nucleic Acids Res. 34:2878‐2886.
   Limbach, P.A., Crain, P.F., Pomerantz, S.C., and McCloskey, J.A. 1995. Appendix 1: Structures of modified nucleosides. In tRNA: Structure, Biosynthesis and Function. (D. Söll and U. Raj Bhandary, eds.). American Society for Microbiology, Washington D.C.
   Luyten, I. and Herdewijn, P. 1998. Hybridization properties of base‐modified oligonucleotides within the double and triple helix motif. Eur. J. Med. Chem. 33:515‐576.
   Manoharan, M. 2004. RNA interference and chemically modified small interfering RNAs. Curr. Opin. Chem. Biol. 8:570‐579.
   Martinez, J., Patkaniowska, A., Urlaub, H., Luhrmann, R., and Tuschl, T. 2002. Single‐stranded antisense siRNAs guide target RNA cleavage in RNAi. Cell 110:563‐574.
   Matranga, C., Tomari, Y., Shin, C., Bartel, D.P., and Zamore, P.D. 2005. Passenger‐strand cleavage facilitates assembly of siRNA into Ago2‐containing RNAi enzyme complexes. Cell. 123:607‐620.
   McSwinggen, J. and Beigelman, L. 2003. RNA interference mediated treatment of Alzheimer's disease using short interfering nucleic acids (siNA). US Patent WO 03/070895.
   Nawrot, B. and Sipa, K. 2006. Chemical and structural diversity of siRNA molecules, Curr. Top. Med. Chem. 6:913‐925.
   Nishimura, S. and Watanabe, K. 2006. The discovery of modified nucleosides from the early days to the present: a personal perspective. J. Biosci. 31:465‐475.
   Nykanen, A., Haley, B., and Zamore, P.D. 2001. ATP requirements and small interfering RNA structure in the RNA interference pathway. Cell 107:309‐321.
   Ogilvie, K.K., Beaucage, S.L., Schifman, A.L., Theriault, N.Y., and Sadana, K.J. 1978. The synthesis of oligoribonucleotides. II. The use of silyl protecting groups in nucleoside and nucleotide chemistry. VIII. Can. J. Chem. 56:2768‐2780.
   Okamoto, I., Shohda, K., Seio, K., and Sekine, M. 2003. A new route to 2′‐O‐alkyl‐2‐thiouridine derivatives via 4‐protection of uracil base and hybridization properties of oligonucleotides incorporating these modified nucleoside derivatives. J. Org. Chem. 68:9971‐9982.
   Parrish, S., Fleenor, J., Xu, S., Mello, C., and Fire, A. 2000. Functional anatomy of a dsRNA trigger: Differential requirement for the two trigger strands in RNA interference. Mol. Cell 6:1077‐1087.
   Rand, T.A., Petersen, S., Du, F., and Wang, X. 2005. Argonaute2 cleaves the anti‐guide strand of siRNA during RISC activation. Cell 123:621‐629.
   Rozenski, J., Crain, P.F., and McCloskey, J.A. 1999. The RNA Modification Database: 1999 update. Nucleic Acids Res. 27:196‐197.
   Schwarz, D.S., Hutvagner, G., Du, T., Xu, Z., Aronin, N., and Zamore, P.D. 2003. Asymmetry in the assembly of the RNAi enzyme complex. Cell 115:199‐208.
   Shohda, K., Okamoto, I., Wada, T., Seio, K., and Sekine, M. 2000. Synthesis and properties of 2′‐O‐methyl‐2‐thiouridine and Oligoribonucleotides containing 2′‐O‐methyl‐2‐thiouridine. Bioorg. Med. Chem. Lett. 10:1795‐1798.
   Sipa, K., Sochacka, E., Kazmierczak‐Baranska, J., Maszewska, M., Janicka, M., Nowak, G., and Nawrot, B. 2007. Effect of base modifications on structure, thermodynamic stability and gene silencing activity of short interfering RNA. RNA 13:1301‐1316.
   Smith, M., Rammler, D.H., Goldberg, I.H., and Khorana, H.G. 1962. Studies on polynucleotides. XVI. Specific synthesis of C3′‐C5′ internucleotide linkage. Synthesis of uridyl‐(3′‐5′)‐uridine and uridyl‐(3′‐5′)‐adenosine. J. Am. Chem. Soc. 84:430‐440.
   Sochacka, E. 2001. Efficient assessment of modified nucleoside stability under conditions of automated oligonucleotide synthesis: characterization of the oxidation and oxidative desulfurization of 2‐thiouridine. Nucleosides Nucleotides Nucleic Acids 20:1871‐1879.
   Soutschek, J., Akinc, A., Bramlage, B., Charisse, K., Constein, R., Donoghue, M., Elbashir, S., Geick, A., Hadwiger, P., Harborth, J., John, M., Kesavan, V., Lavine, G., Pandey, R.K., Racie, T., Rajeev, K.G., Rohl, I., Toudjarska, I., Wang, G., Wuschko, S., Bumcrot, D., Koteliansky, V., Limmer, S., Manoharan, M., and Vornlocher, H.P. 2004. Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs. Nature 432:173‐178.
   Stuart, J.W., Basti, M.M., Smith, W., Forrest, B., Guenther, R., Sierzputowska‐Gracz, H., Nawrot, B., Malkiewicz, A., and Agris, P.F. 1996. Structure of Trinucleotide Dp(acp)3UpA with Coordinated Mg+2 Demonstrates that Modified Nucleosides Contribute to Regional Conformations of tRNA. Nucleosides Nucleotides 15:1009‐1028.
   Stuart, J.W., Gdaniec, Z., Guenther, R., Marszałek, M., Sochacka, E., Malkiewicz, A., and Agris, P.F. 2000. Functional anticodon architecture of human tRNALys3 includes disruption of intraloop hydrogen bonding by the naturally occurring amino acid modification, t6A. Biochemistry 39:13390‐13395.
   Sundaram, M., Durant, P.C., and Davis, D.R. 2000. Hypermodified nucleosides in the anticodon of tRNALys stabilize a canonical U‐turn structure. Biochemistry 39:12575‐12584.
   Tang, G., Reinhart, B.J., Bartel, D.P., and Zamore, P.D. 2003. A biochemical framework for RNA silencing in plants. Genes Dev. 17:49‐63.
   Testa, S.M., Disney, M.D., Turner, D.H., and Kierzek, R. 1999. Thermodynamics of RNA‐RNA duplexes with 2‐ or 4‐thiouridines: Implications for antisense design and targeting a group I intron. Biochemistry 38:16655‐16662.
   Uprichard, S.L. 2005. The therapeutic potential of RNA interference. FEBS Lett. 579:5996‐6007.
   Vaught, J.D., Dewey, T., and Eaton, B.E. 2004. T7 RNA polymerase transcription with 5‐position modified UTP derivatives. J. Am. Chem. Soc. 126:11231‐11337.
   Venkatesan, N., Kim, S.J., and Kim, B.H. 2003. Novel phosphoramidite building blocks in synthesis and applications towards modified oligonucleotides. Curr. Med. Chem. 10:1973‐1991.
   Verma, S. and Eckstein, F. 1998. Modified oligonucleotides: Synthesis and strategy for users. Annu. Rev. Biochem. 67:99‐134.
   Verma, S., Jager, S., Thum, O., and Famulok, M. 2003. Functional tuning of nucleic acids by chemical modifications: Tailored oligonucleotides as drug, devices, and diagnostics. Chem. Rec. 3:61‐60.
   Vorbruggen, H. and Strehlke, P. 1973. Eine einfache Synthese von 2‐Thiopyrimidin‐nucleosiden. Chem. Ber. 106:3039‐3061.
   Westhof, E., Dumas, P., and Moras, D. 1988. Restrained refinement of two crystalline forms of yeast aspartic acid and phenylalanine transfer RNA crystals. Acta Cryst. A. 44:112‐123.
   Yarian, C., Marszałek, M., Sochacka, E., Malkiewicz, A., Guenther, R., Miskiewicz, A., and Agris, P.F. 2000. Modified nucleoside dependent Watson‐Crick and wobble codon binding by tRNALys (UUU) species. Biochemistry 39:13396‐13404.
   Zamore, P.D., Tuschl, T., Sharp, P.A., and Bartel, D.P. 2000. RNAi: Double‐stranded RNA directs the ATP‐dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101:25‐33.
PDF or HTML at Wiley Online Library