Molecular Diversity

, Volume 15, Issue 3, pp 677–686 | Cite as

Synthesis and properties of small interfering RNA duplexes carrying 5-ethyluridine residues

  • Montserrat Terrazas
  • Ramon Eritja
Full-Length Paper


Oligoribonucleotides carrying 5-ethyluridine units were prepared using solid-phase phosphoramidite chemistry. The introduction of the tert-butyldimethylsilyl group at the 2′-OH position proceeded in good yield and very high 2′-regioselectivity. RNA duplexes carrying 5-ethyluridine either at the sense or the guide strands display RNAi activity comparable to or slightly better than that of unmodified RNA duplexes. Gene suppression experiments using luciferase targets in SH-SY5Y cells show that the ethyl group is generally well accepted at all positions although a small decrease in RNA interference activity is observed when one 5-ethylU residue is incorporated in the 3′ overhangs.


SiRNA RNA interference 5-Ethyluridine Dual luciferase assay Oligonucleotide synthesis Solid-phase 



Guide (antisense) strand








Dulbecco’s modified Eagle medium




Ethyl acetate


Fetal bovine serum


4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid




Small interfering RNA


Sense strand


Tetrabutylammonium fluoride




Triethylammonium acetate


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

Supplementary material

11030_2010_9290_MOESM1_ESM.pdf (2.1 mb)
ESM 1 (PDF 2125 kb)


  1. 1.
    Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391: 806–811. doi: 10.1038/35888 PubMedCrossRefGoogle Scholar
  2. 2.
    Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411: 494–498. doi: 10.1038/35078107 PubMedCrossRefGoogle Scholar
  3. 3.
    Behlke MA (2008) Chemical modification of siRNA for in vivo use. Oligonucleotides 18: 305–320. doi: 10.1089/oli.2008.0164 PubMedCrossRefGoogle Scholar
  4. 4.
    Watts JK, Deleavey GF, Damha MJ (2008) Chemically modified siRNA: tools and applications. Drug Discov Today 13: 842–855. doi: 10.1016/j.drudis.2008.05.007 PubMedCrossRefGoogle Scholar
  5. 5.
    Matranga C, Tomari Y, Shin C, Bartel DP, Zamore PD (2005) Passenger-strand cleavage facilitates assembly of siRNA into Ago-2 containing RNAi enzyme complexes. Cell 123: 607–620. doi: 10.1016/j.cell.2005.08.044 PubMedCrossRefGoogle Scholar
  6. 6.
    Bramsen JB, Laursen MB, Nielsen AF, Hansen TB, Bus C, Lankjaer N, Babu BR, Hojland T, Abramov M, van Aerschot A, Odadzic D, Smicius R, Haas J, Andree C, Barman J, Wenska M, Srivastava P, Zhou C, Honcharenko D, Hess S, Múller E, Bobkov GV, Mikhailov SN, Fava E, Meyer TF, Chatopadhyaya J, Zerial M, Engels JW, Herdewijn P, Wengel J, Kjems J. (2009) A large-scale chemical modification screen identifies design rules to generate siRNAs with high stability and low toxicity. Nucleic Acids Res 37: 2867–2881. doi: 10.1093/nar/gkp106 PubMedCrossRefGoogle Scholar
  7. 7.
    de Fougerolles A, Vornlocher HP, Maraganore J, Lieberman J (2007) Interfering with disease: a progress report on siRNA-based therapeutics. Nat Rev Drug Discov 6: 443–453. doi: 10.1038/nrd2310 PubMedCrossRefGoogle Scholar
  8. 8.
    de Martimprey H, Vauthier C, Malvy C, Couvreur P (2009) Polymer nanocarriers for the delivery of small fragments of nucleic acids: oligonucleotides and siRNA. Eur J Pharm Biopharm 71: 490–504. doi: 10.1016/j.ejpb.2008.09.024 PubMedCrossRefGoogle Scholar
  9. 9.
    Soutschek J, Akinc A, Bramlage B, Charisse K, Constien R, Donoghue M, Elbashir S, Geick A, Hadwiger P, Harborth J, John M, Kesavan V, Lavine G, Pandey RK, Racie T, Rajeev KG, Röhl I, Toudjarska I, Wang G, Wuschko S, Bumcrot D, Koteliansky V, Limmer S, Manoharan M, Vornlocher HP (2004) Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs. Nature 432: 173–178. doi: 10.1038/nature03121 PubMedCrossRefGoogle Scholar
  10. 10.
    Said Hassane F, Saleh AF, Abes R, Gait MJ, Lebleu B (2010) Cell penetrating peptides: overview and applications to the delivery of oligonucleotides. Cell Mol Life Sci 67:715–726. doi: 10.1007/s00018-009-0186-0 Google Scholar
  11. 11.
    Giljohann DA, Seferos DS, Prigodich AE, Patel PC, Mirkin CA (2009) Gene regulation with polyvalent siRNA-nanoparticle conjugates. J Am Chem Soc 131: 2072–2073. doi: 10.1021/ja808719p PubMedCrossRefGoogle Scholar
  12. 12.
    Chiu YL, Rana TM (2003) siRNA function in RNAi: a chemical modification analysis. RNA 9: 1034–1048. doi: 10.1261/rna.5103703 PubMedCrossRefGoogle Scholar
  13. 13.
    Parrish S, Fleenor J, Xu SQ, Mello C, Fire A (2000) Functional anatomy of a dsRNA trigger: differential requirement for the two trigger strands in RNA interference. Mol Cell 6: 1077–1087. doi: 10.1016/S1097-2765(00)00106-4 PubMedCrossRefGoogle Scholar
  14. 14.
    Sipa K, Sochacka E, Kazmierczak-Baranska J, Maszewska M, Janicka M, Nowak G, Nawrot B (2007) Effect of base modifications on structure, thermodynamic stability, and gene silencing activity of short interfering RNA. RNA 13: 1301–1316. doi: 10.1261/rna.538907 PubMedCrossRefGoogle Scholar
  15. 15.
    Somoza A, Chelliserrykattil J, Kool ET (2006) The roles of hydrogen bonding and sterics in RNA interference. Angew Chem Int Edn 45: 4994–4997. doi: 10.1002/anie.200601311 CrossRefGoogle Scholar
  16. 16.
    Xia J, Noronha A, Toudjarska I, Li F, Akinc A, Braich R, Frank-Kamenetsky M, Rajeev KG, Egli M, Manoharan M (2006) Gene silencing activity of siRNAs with a ribo-difluorotoluyl nucleotide. ACS Chem Biol 1: 176–183. doi: 10.1021/cb600063p PubMedCrossRefGoogle Scholar
  17. 17.
    Somoza A, Silverman AP, Miller RM, Chelliserrykattil J, Kool ET (2008) Steric Effects in RNA interference: probing the influence of nucleobase size and shape. Chem Eur J 14: 7978–7987. doi: 10.1002/chem.200800837 CrossRefGoogle Scholar
  18. 18.
    Terrazas M, Kool ET (2009) RNA major groove modifications improve siRNA stability and biological activity. Nucleic Acids Res 37: 346–353. doi: 10.1093/nar/gkn958 PubMedCrossRefGoogle Scholar
  19. 19.
    Bergstrom DE, Ruth JL (1976) Synthesis of C-5 substituted pyrimidine nucleosides via organopalladium intermediates. J Am Chem Soc 98: 1587–1589. doi: 10.1021/ja00422a056 PubMedCrossRefGoogle Scholar
  20. 20.
    Bergstrom DE, Ogawa MK (1978) C-5 Substituted pyrimidine nucleosides. 2. Synthesis via olefin coupling to organopalladium intermediates derived from uridine and 2′-deoxyuridine. J Am Chem Soc 100: 8106–8112. doi: 10.1021/ja00494a014 CrossRefGoogle Scholar
  21. 21.
    Shapira J (1962) Synthesis of 5-ethyluridine, a model 5-alkylsubstituted pyrimidine nucleoside. J Org Chem 27: 1918–1919. doi: 10.1021/jo01052a528 CrossRefGoogle Scholar
  22. 22.
    Niedballa U, Vorbrüggen H (1974) Synthesis of nucleosides, 9, General synthesis of N-glycosides. I. Synthesis of pyrimidine nucleosides. J Org Chem 39: 3654–3660. doi: 10.1021/jo00939a008 PubMedCrossRefGoogle Scholar
  23. 23.
    Herdewijn P, Kerremans L, Wigerinck P, Vandendriessche F, van Aerschot A (1991) Synthesis of thymidine from 5-iodo-2′-deoxyuridine. Tetrahedron Lett 32: 4397–4400. doi: 10.1016/S0040-4039(00)92180-4 CrossRefGoogle Scholar
  24. 24.
    Hakimelahi GH, Proba ZA, Ogilvie KK (1982) New catalysts and procedures for the dimethoxytritylation and selective silylation of ribonucleosides. Can J Chem 60: 1106–1113. doi: 10.1139/v82-165 CrossRefGoogle Scholar
  25. 25.
    Harborth J, Elbashir SM, Vandenburgh K, Manninga H, Scaringe SA, Weber K, Tuschl T (2003) Sequence, chemical, and structural variation of small interfering RNAs and short hairpin RNAs and the effect on mammalian gene silencing. Antisense Nucleic A 13: 83–105. doi: 10.1089/108729003321629638 CrossRefGoogle Scholar
  26. 26.
    Hayakawa T, Ono A, Ueda T (1988) Synthesis of decadeoxyribonucleotides containing 5-modified uracils and their interactions with restriction endonucleases BglII, Sau3AI and MboI. Nucleic Acids Res 16: 4761–4776. doi: 10.1093/nar/16.11.4761 PubMedCrossRefGoogle Scholar
  27. 27.
    Kypr J, Sági J, Szakonyi E, Ebinger K, Penazová H, Chládkova J, Vorlícková M (1994) Thymine methyl groups stabilize the putative A-form of the synthetic DNA poly(amino 2 dA-dT). Biochemistry-US 33: 3801–3806. doi: 10.1021/bi00179a003 CrossRefGoogle Scholar
  28. 28.
    Marzabal S, DuBois S, Thielking V, Cano A, Eritja R, Guschlbauer W (1995) Dam methylase from Escherichia coli: kinetic studies using modified DNA oligomers: hemimethylated substrates. Nucleic Acids Res 23: 3648–3655. doi: 10.1093/nar/23.18.3648 PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  1. 1.Institute for Research in Biomedicine (IRB Barcelona)Institute for Advanced Chemistry of Catalonia (IQAC-CSIC), Networking Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN)BarcelonaSpain

Personalised recommendations