Solid‐Phase Synthesis of Oligodeoxynucleotide Analogs Containing Phosphorodithioate Linkages

Xianbin Yang1

1 AM Biotechnologies, LLC, Houston, Texas
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.71
DOI:  10.1002/cpnc.13
Online Posting Date:  September, 2016
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The oligodeoxynucleotide phosphorodithioate modification (PS2‐ODN) uses two sulfur atoms to replace two non‐bridging oxygen atoms at an internucleotide phosphordiester backbone linkage. Like a natural phosphodiester ODN backbone linkage, a PS2‐modified backbone linkage is achiral at phosphorus. PS2‐ODNs are highly stable to nucleases and numerous in vitro assays have demonstrated their biological activity. For example, PS2‐ODNs activated RNase H in vitro, strongly inhibited human immunodeficiency virus (HIV) reverse transcriptase, induced B‐cell proliferation and differentiation, and bound to protein targets in the form of PS2‐aptamers (thioaptamers). Thus, the interest in and promise of PS2‐ODNs has spawned a variety of strategies for synthesizing, isolating, and characterizing this compounds. ODN‐thiophosphoramidite monomers are commercially available from either AM Biotechnologies or Glen Research and this unit describes an effective methodology for solid‐phase synthesis, deprotection, and purification of ODNs having PS2 internucleotide linkages. © 2016 by John Wiley & Sons, Inc.

Keywords: solid‐phase synthesis; phosphorodithioate oligodeoxynucleotide; PS2‐ODN; sulfur‐modified oligonucleotide; thiophosphoramidite

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Table of Contents

  • Introduction
  • Basic Protocol 1: Solid‐Phase Assembly of Protected ODN‐Thiophosphoramidites
  • Basic Protocol 2: Deprotection and Purification of PS2‐ODNs
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Solid‐Phase Assembly of Protected ODN‐Thiophosphoramidites

  • ODN‐thiophosphoramidites or DNA‐thiophosphoramidites (AM Biotechnologies or Glen Research):
    • dA‐thiophosphoramidite
    • dC‐thiophosphoramidite or Ac‐dC‐thiophosphoramidite
    • dG‐thiophosphoramidite
    • dT‐thiophosphoramidite
  • Normal phosphoramidites (Glen Research):
    • dA‐CE phosphoramidite
    • dC‐CE phosphoramidite or Ac‐dC‐CE Phosphoramidite
    • dG‐CE Phosphoramidite
    • dT‐CE Phosphoramidite
  • Argon gas
  • Activator:
    • 0.45 M tetrazole in acetonitrile (Glen Research)
    • 0.25 M DCI (4,5‐dicyanoimidazole) in acetonitrile (Sigma‐Aldrich)
    • 0.25 M activator‐42 (Sigma‐Aldrich)
  • Diluent: acetonitrile, anhydrous (Glen Research) containing 10% anhydrous dichloromethane (DCM)
  • Helium gas, anhydrous
  • Cap A: tetrahydrofuran/acetic anhydride (THF/ Ac 2O) (Glen Research)
  • Cap B: 10% 1‐methyllimidazole in tetrahydrofuran/ pyridine (Glen Research)
  • Oxidizing solution: 0.02 M I 2 in THF/H 2O/pyridine (Glen Research)
  • Deblocking mix: 3% trichloroacetic acid in dichloromethane (Glen Research)
  • Sulfurization reagent: 3‐ethoxy‐1,2,4‐dithiazolidine‐5‐one (EDITH), 0.2 M (163 mg in 20 mL acetonitrile), molecular weight: 163.22 (Carbosynth Limited)
  • Synthesizer vials with caps
  • Vacuum desiccators
  • LCA‐CPG column (Glen Research)
  • Expedite 8909, Perseptive Biosystem with trityl monitor

Basic Protocol 2: Deprotection and Purification of PS2‐ODNs

  • Argon gas
  • Fully protected ODNs attached to the solid support of a synthesis column (see protocol 1)
  • Anhydrous ethanol (Sigma‐Aldrich)
  • 28% ammonium hydroxide or concentrated ammonium hydroxide (Sigma‐Aldrich)
  • DL‐Dithiothreitol (Sigma‐Aldrich)
  • Ammonium acetate (HPLC grade, Fluka)
  • Deionized water
  • 24% denaturing polyacrylamide gel
  • Loading buffer (Promega, cat. no. G1881)
  • Acetonitrile (HPLC grade, TEDIA)
  • Trizma hydrochloride (Sigma‐Aldrich), pH 8
  • EDTA (Sigma‐Aldrich)
  • Sodium hydroxide NaCl; Sigma‐Aldrich)
  • Monobeads
  • 4.0‐mL sealable vial
  • 55°C incubator
  • 15‐mL conical tubes
  • −70°C freezer
  • Lyophilizer
  • 1000‐μL UV cuvettes
  • Spectrophotometer
  • 0.75‐mm analytical gel plate cassettes
  • Electrophoresis apparatus
  • Plastic wrap
  • TLC Merck silica plate
  • Hand‐held 254‐nm UV lamp
  • Camera with a UV filter
  • Razor blades
  • Spatula
  • Platform shaker
  • 0.22‐μm hydrophilic fluid filter
  • SepPak C18 cartridges
  • DIONEX DNAPac PA‐100 4 × 250 mm analytical column (cat. no. 043010)
  • Mono‐Q columns
  • A divinyl benzene/polystyrene copolymer reverse‐phase column (Hamilton PRP‐1): Part No. 79426, Flow = 2 ml/min or Part No. 79425, Flow = 1 mL/min
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Literature Cited

  Brill, W.K.D., Nielsen, J., and Caruthers, M.H. 1988. Synthesis of dinucleoside phosphorodithioates via thioamidites. Tetrahedron Lett. 29:5517‐5520. doi:10.1016/S0040‐4039(00)80801‐1
  Cheng, X., DeLong, R.K., Wickstrom, E., Kligshteyn, M., Demirdji, S.H., Caruthers, M.H., and Juliano, R.L. 1997. Interactions between single‐stranded DNA binding protein and oligonucleotide analogs with different backbone chemistries. J. Mol. Recognit. 10:101‐107. doi: 10.1002/(SICI)1099‐1352(199703/04)10:2<101::AID‐JMR344>3.0.CO;2‐4.
  Cummins, L., Graff, D., Beaton, G., Marshall, W.S., and Caruthers, M.H. 1996. Biochemical and physicochemical properties of phosphorodithioate DNA. Biochemistry 35:8734‐8741.
  Dahl, B.H., Bjergarde, K., Sommer, V.B., and Dahi, O. 1989. Deoxynucleoside phosphorodithioates. Acta Chem. Scand. 43:896‐901.
  Eckstein, F. 2000. Phosphorothioate oligodeoxynucleotides: What is their origin and what is unique about them? Antisense Nucleic Acid Drug Dev. 10:117‐121. doi: 10.1089/oli.1.2000.10.117.
  Ellington, A.D. and Szostak, J.W. 1990. In vitro selection of RNA molecules that bind specific ligands. Nature 346:818‐822. doi: 10.1038/346818a0.
  Farschtschi, N. and Gorenstein, D.G. 1988. Preparation of a deoxynucleoside thiophosphoramidite intermediate in the synthesis of nucleoside Phosphorodithioates. Tetrahedron Lett. 29:6843‐6846. doi: 10.1016/S0040‐4039(00)88455‐5.
  Fennewald, S.M., Scott, E.P., Zhang, L., Yang, X., Aronson, J.F., Gorenstein, D.G., Luxon, B.A., Shope, R.E., Beasley, D.W., Barrett, A.D., and Herzog, N.K. 2007. Thioaptamer decoy targeting of AP‐1 proteins influences cytokine expression and the outcome of arenavirus infections. J. Gen. Virol. 88:981‐990.
  Ferguson, M.R., Rojo, D.R., Somasunderam, A., Thiviyanathan, V., Ridley, B.D., Yang, X., and Gorenstein, D.G. 2006. Delivery of double‐stranded DNA thioaptamers into HIV‐1 infected cells for antiviral activity. Biochem. Biophys. Res. Commun. 344:792‐797. doi: 10.1016/j.bbrc.2006.03.201.
  Guga, P. and Stec, W.J. 2003. Synthesis of phosphorothioate oligonucleotides with stereodefined phosphorothioate linkages. Curr. Protoc. Nucleic Acid Chem. 14:4.17.1–4.17.28. doi: 10.1002/0471142700.nc0417s14.
  Hecht, A.H., Sommer, G.J., Durland, R.H., Yang, X., Singh, A.K., and Hatch, A.V. 2010. Aptamers as affinity reagents in an integrated electrophoretic lab‐on‐a‐chip platform. Anal. Chem. 82:88‐13‐8820. doi: 10.1021/ac101106m.
  Krieg, A.M., Matson, S., and Fisher, E. 1996. Oligodeoxynucleotide modifications determine the magnitude of B cell stimulation by CpG motifs. Antisense Nucleic Acid Drug Dev. 6:133‐139. doi: 10.1089/oli.1.1996.6.133.
  Marshall, W.S. and Caruthers, M.H. 1993. Phosphorodithioate DNA as a potential therapeutic drug. Science 259:1564‐1570. doi: 10.1126/science.7681216.
  Marshall, W.S., Beaton, G., Stein, C.A., Matsukura, M., and Caruthers, M.H. 1992. Inhibition of human immunodeficiency virus activity by phosphorodithioate oligodeoxycytidine. Proc. Natl. Acad. Sci. USA 89:6265‐6269.
  Nielsen, J., Brill, W.K.D., and Caruthers, M.H. 1988. Synthesis and characterization of dinucleoside phosphorodithioates. Tetrahedron Lett. 29:2911‐2914. doi: 10.1016/0040‐4039(88)85045‐7.
  Sierant, M., Yang, X., Janicka, M., Li, N., Martinez, C., Hassell, T., and Nawrot, B. 2011. SiRNA with phosphorodithioate modification. Collection Symposium Series, Cesky Krumlov. June 5 ‐10.
  Stec, W.J. and Wilk, A. 1994. Stereocontrolled synthesis of oligonucleoside phosphorothioates. Angew Chem. Int. Ed. Engl. 33:709‐722. doi: 10.1002/anie.199407091.
  Stec, W.J., Grajkowski, A., Koziolkiewicz, M., and Uznanski, B. 1991. Novel route to oligo(deoxyribonucleoside phosphorothioates). Stereocontrolled synthesis of P‐chiral oligo(deoxyribonucleoside phosphorothioates). Nucleic Acids Res. 19:5883‐5888. doi: 10.1093/nar/19.21.5883.
  Stein, C.A. and Cheng, Y.C. 1993. Antisense oligonucleotides as therapeutic agents–is the bullet really magical? Science 261:1004‐1012. doi: 10.1126/science.8351515.
  Stephenson, M.L. and Zamecnik, P.C. 1978. Inhibition of Rous sarcoma viral RNA translation by a specific oligodeoxyribonucleotide. Proc. Natl. Acad. Sci. USA 75:285‐288. doi: 10.1073/pnas.75.1.285.
  Tonkinson, J.L., Guvakova, M., Khaled, Z., Lee, J., Yakubov, L., Marshall, W.S., Caruthers, M.H., and Stein, C.A. 1994. Cellular pharmacology and protein binding of phosphoromonothioate and phosphorodithioate oligodeoxynucleotides: A comparative study. Antisense Res. Dev. 4:269‐278.
  Tuerk, C. and Gold, L. 1990. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249:505‐510. doi: 10.1126/science.2200121.
  Uhlmann, E. and Peyman, A. 1990. Antisense oligonucleotides: A new therapeutic principle. Chem. Rev. 90:543‐585. doi: 10.1021/cr00102a001.
  Vaughn, J.P., Stekler, J., Demirdji, S., Mills, J.K., Caruthers, M.H., Iglehart, J.D., and Marks, J.R. 1996. Inhibition of the erbB‐2 tyrosine kinase receptor in breast cancer cells by phosphoromonothioate and phosphorodithioate antisense oligonucleotides. Nucleic Acids Res. 24:4558‐4564. doi: 10.1093/nar/24.22.4558.
  Wada, T., Fujiwara, S., Sato, T., Oka, N., and Saigo, K. 2004. Stereocontrolled synthesis of phosphorothioate RNA by the oxazaphospholidine approach. Nucleic Acids Symp. Ser. (Oxf) 3:109‐110.doi: 10.1093/nass/48.1.57.
  Wang, H., Yang, X., Bowick, G.C., Herzog, N.K., Luxon, B.A., Lomas, L.O., and Gorenstein, D.G. 2006. Identification of proteins bound to a thioaptamer probe on a proteomics array. Biochem. Biophys. Res. Commun. 347:586‐593. doi: 10.1016/j.bbrc.2006.06.132.
  Wiesler, W.T. and Caruthers, M.H. 1996. Synthesis of phosphorodithioate DNA via sulfur‐linked, base‐labile protecting groups. J. Org. Chem. 61:4272‐4281. doi: 10.1021/jo960274y.
  Wiesler, W.T., Marshall, W.S., and Caruthers, M.H. 1993. Synthesis and purification of phosphorodithioate DNA. Methods Mol. Biol. 20:191‐206.
  Wilk, A. and Stec, W.J. 1995. Analysis of oligo(deoxynucleoside phosphorothioate)s and their diastereomeric composition. Nucleic Acids Res. 23:530‐534. doi: 10.1093/nar/23.3.530.
  Wu, S.Y., Yang, X., Gharpure, K.M., Hatakeyama, H., Egli, M., McGuire, M.H., Nagaraja, A.S., Miyake, T.M., Rupaimoole, R., Pecot, C.V., Taylor, M., Pradeep, S., Sierant, M., Rodriguez‐Aguayo, C., Choi, H.J., Previs, R.A., Armaiz‐Pena, G.N., Huang, L., Martinez, C., Hassell, T., Ivan, C., Sehgal, V., Singhania, R., Han, H.D., Su, C., Kim, J.H., Dalton, H.J., Kovvali, C., Keyomarsi, K., McMillan, N.A., Overwijk, W.W., Liu, J., Lee, J.S., Baggerly, K.A., Lopez‐Berestein, G., Ram, P.T., Nawrot, B., and Sood, A.K. 2014. 2′‐OMe‐phosphorodithioate‐modified siRNAs show increased loading into the RISC complex and enhanced anti‐tumour activity. Nat. Commun. 5:3459. doi: 10.1038/ncomms4459.
  Yamamoto, T., Nakatani, M., Narukawa, K., and Obika, S. 2011. Antisense drug discovery and development. Future Med. Chem. 3:339‐365. doi: 10.4155/fmc.11.2.
  Yang, X. and Gorenstein, D.G. 2004. Progress in thioaptamer development. Curr. Drug Targets 5:705‐715. doi: 10.2174/1389450043345074.
  Yang, X. and Mierzejewski, E. 2010. Synthesis of nucleoside and oligonucleoside dithiophosphates. New J. Chem. 34:805‐819. doi: 10.1039/b9nj00618d.
  Yang, X., Misiura, K., and Stec, W.J. 1999c. Reactivity of nucleoside 5′‐O‐phosphates,‐phosphorothioates, methanephosphonates, and ‐methanephosphonothioates toward activated xylonucleosides. Heteroatom Chem. 10:91‐104. doi: 10.1002/(SICI)1098‐1071(1999)10:2%3c91::AID‐HC2%3e3.0.CO;2‐Y.
  Yang, X., Li, N., and Gorenstein, D.G. 2011. Strategies for the discovery of therapeutic aptamers. Expert Opin. Drug Discov. 6:75‐87. doi: 10.1517/17460441.2011.537321.
  Yang, X., Misiura, K., Sochacki, M., and Stec, W. J. 1997. Deoxyxylothymidine 3′‐O‐phosphorothioates: Synthesis, stereochemistry and stereocontrolled incorporation into oligothymidylates. Bioorg. Med. Chem. Lett. 7:2651‐2656. doi: 10.1016/S0960‐894X(97)10040‐3.
  Yang, X., Hodge, R.P., Luxon, B.A., Shope, R., and Gorenstein, D.G. 2002b. Separation of synthetic oligonucleotide dithioates from monothiophosphate impurities by anion‐exchange chromatography on a mono‐q column. Anal. Biochem. 306:92‐99. doi: 10.1006/abio.2001.5694.
  Yang, X., Fennewald, S., Luxon, B.A., Aronson, J., Herzog, N.K., and Gorenstein, D.G. 1999. Aptamers containing thymidine 3′O‐phosphorodithioates: Synthesis and binding to nuclear factor‐kappaB. Bioorg. Med. Chem. Lett. 9:3357‐3362. doi: 10.1016/S0960‐894X(99)00600‐9.
  Yang, X.B., Sierzchala, A., Misiura, K., Niewiarowski, W., Sochacki, M., Stec, W.J., and Wieczorek, M.W. 1998. The First Stereocontrolled Solid‐Phase Synthesis of Di‐, Tri‐, and Tetra[adenosine (2′,5′) phosphorothioate]s. J. Org. Chem. 63:7097‐7100. doi: 10.1021/jo980522l.
  Yang, X., Sierant, M., Janicka, M., Peczek, L., Martinez, C., Hassell, T., Li, N., Li, X., Wang, T., and Nawrot, B. 2012. Gene Silencing activity of siRNA molecules containing phosphorodithioate substitutions. ACS Chem. Biol. 7:1214‐1220. doi: 10.1021/cb300078e.
  Yang, X., Bassett, S.E., Li, X., Luxon, B.A., Herzog, N.K., Shope, R.E., Aronson, J., Prow, T.W., Leary, J.F., Kirby, R., Ellington, A.D., and Gorenstein, D.G. 2002. Construction and selection of bead‐bound combinatorial oligonucleoside phosphorothioate and phosphorodithioate aptamer libraries designed for rapid PCR‐based sequencing. Nucleic Acids Res. 30:e132. doi: 10.1093/nar/gnf132.
  Yang, X., Wang, H., Beasley, D.W., Volk, D.E., Zhao, X., Luxon, B.A., Lomas, L.O., Herzog, N.K., Aronson, J.F., Barrett, A.D., Leary, J.F., and Gorenstein, D.G. 2006. Selection of thioaptamers for diagnostics and therapeutics. Ann. N. Y. Acad. Sci. 1082:116‐119. doi: 10.1196/annals.1348.065.
  Zamecnik, P.C. and Stephenson, M.L. 1978. Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide. Proc. Nat. Acad. Sci. USA 75:280‐284. doi: 10.1073/pnas.75.1.280.
  Zandarashvili, L., Nguyen, D., Anderson, K.M., White, M.A., Gorenstein, D.G., and Iwahara, J. 2015. Entropic enhancement of Protein‐DNA affinity by oxygen‐to‐sulfur substitution in DNA phosphate. Biophys. J. 109:1026‐1037. doi: 10.1016/j.bpj.2015.07.032.
Key References
  Yang and Mierzejewsk, 2010. See above.
  This reference summarizes most of the methods (if not all) for synthesis of PS2‐ODNs, as well as the synthesis of PS2‐ODN building block.
  Wiesler and Caruthers, 1996. See above.
  This reference describes the detailed procedure for synthesis of ODN‐thiophosphoramidites and their application.
  Yang et al., 2002b. See above.
  This reference describes the detailed method to purify the PS2‐ODNs.
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