Solid‐Phase Synthesis of RNA Analogs Containing Phosphorodithioate Linkages

Xianbin Yang1

1 AM Biotechnologies, Houston, Texas
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.77
DOI:  10.1002/cpnc.40
Online Posting Date:  September, 2017
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The oligoribonucleotide phosphorodithioate (PS2‐RNA) modification uses two sulfur atoms to replace two non‐bridging oxygen atoms at an internucleotide phosphorodiester backbone linkage. Like a natural phosphodiester RNA backbone linkage, a PS2‐modified backbone linkage is achiral at phosphorus. PS2‐RNAs are highly stable to nucleases and several in vitro assays have demonstrated their biological activity. For example, PS2‐RNAs silenced mRNA in vitro and bound to protein targets in the form of PS2‐aptamers (thioaptamers). Thus, the interest in and promise of PS2‐RNAs has drawn attention to synthesizing, isolating, and characterizing these compounds. RNA‐thiophosphoramidite monomers are commercially available from AM Biotechnologies and this unit describes an effective methodology for solid‐phase synthesis, deprotection, and purification of RNAs having PS2 internucleotide linkages. © 2017 by John Wiley & Sons, Inc.

Keywords: phosphorodithioate oligoribonucleotide; PS2‐RNA; solid‐phase synthesis; sulfur‐modified oligonucleotide; thiophosphoramidite

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Table of Contents

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

  • RNA‐thiophosphoramidites (AM Biotechnologies)
    • Bz‐rA‐thiophosphoramidite
    • Ac‐rC‐thiophosphoramidite
    • Ac‐rG‐thiophosphoramidite
    • rU‐thiophosphoramidite
  • Normal RNA phosphoramidites (Glen Research)
    • Bz‐A‐CE phosphoramidite
    • Ac‐C‐CE phosphoramidite
    • Ac‐G‐CE phosphoramidite
    • U‐CE phosphoramidite
  • XX‐RNA‐CPG column (Glen Research)
  • Argon gas, anhydrous
  • Acetonitrile, anhydrous (Glen Research)
  • 10% (v/v) anhydrous dichloromethane in anhydrous acetonitrile
  • 0.25 M Activator 42 (Sigma‐Aldrich)
  • Cap A mix, tetrahydrofuran/acetic anhydride (THF/Ac 2O; Glen Research)
  • Cap B mix, 10% 1‐methylimidazole in tetrahydrofuran/pyridine (Glen Research)
  • 0.02 M I 2 in THF/H 2O/pyridine (oxidizing solution; Glen Research)
  • 3% trichloroacetic acid (TCA) in dichloromethane (deblocking mix; Glen Research)
  • 0.2 M 3‐ethoxy‐1,2,4‐dithiazolidine‐5‐one (EDITH; MW 163.22) in acetonitrile (sulfurization reagent; 163 mg in 20 mL acetonitrile, Carbosynth Limited)
  • Wash A (acetonitrile, anhydrous; Glen Research)
  • Synthesizer vials with caps
  • Vacuum desiccators
  • Expedite 8909 (Perseptive Biosystem) with trityl monitor

Basic Protocol 2: Deprotection and Purification of PS2‐RNAs

  • Fully protected PS2‐RNAs attached to solid support of a synthesis column (see protocol 1)
  • Argon gas, anhydrous
  • Anhydrous ethanol (Sigma‐Aldrich)
  • Acetonitrile (HPLC grade, TEDIA)
  • Mobile phase A (1 mM EDTA, 25 mM Tris·HCl, pH 8)
  • Mobile phase B (1 mM EDTA, 25 mM Tris·HCl, 1 M NaCl, pH 8)
  • Sodium hydroxide (for adjusting pH of mobile phases; Sigma‐Aldrich)
  • >28% ammonium hydroxide or concentrated ammonium hydroxide (Sigma‐Aldrich)
  • 41% methyl amine (Fluka, cat. no. 65580)
  • Triethylamine (TEA; Sigma‐Aldrich)
  • DL‐Dithiothreitol (DDT; Sigma‐Aldrich)
  • Triethylamine trihydrofluoride, 98% (Sigma‐Aldrich, cat. no. 344648)
  • N,N‐Dimethylformamide (DMF; Sigma‐Aldrich)
  • Ammonium acetate (HPLC grade, Fluka)
  • Diethylpyrocarbonate (DEPC; Sigma‐Aldrich)
  • Loading buffer (50% glycerol, 1 mM EDTA, 0.4% bromophenol blue, 1 mg/mL ethidium bromide in DEPC‐treated or nuclease‐free water)
  • Vials, 4 mL and sealable
  • Incubator, 55°C (temperature range: up to 100°C)
  • Sterile syringe filter, 0.2 μm cellulose acetate (VWR, cat. no. 28145‐477)
  • Dionex DNAPac PA‐100 4 × 250‐mm analytical column (Thermo Fisher Scientific, cat. no. 043010)
  • Divinyl benzene/polystyrene copolymer reverse‐phase column (Hamilton PRP‐1, cat. no. 79426, flow rate 2 mL/min or cat. no. 79425, flow rate 1 mL/min)
  • Mono Q column (GE Healthcare Life Sciences)
  • Amicon ultra‐15 centrifugal filter units, 3000 Da cut‐off (Millipore Sigma)
  • Silica TLC plates (Merck)
  • Platform shaker
  • Lyophilizer
  • SepPak C18 cartridges
Additional reagents and equipment for SDS‐PAGE (Gallagher, )
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Literature Cited

  Abeydeera, N. D., Egli, M., Cox, N., Mercier, K., Conde, J. N., Pallan, P. S., … Yang, X. (2016). Evoking picomolar binding in RNA by a single phosphorodithioate linkage. Nucleic Acids Research, 44, 8052–8064. doi: 10.1093/nar/gkw725.
  Behlke, M. A. (2006). Progress towards in vivo use of siRNAs. Molecular Therapy, 13, 644–670. doi: 10.1016/j.ymthe.2006.01.001.
  Blank, M., & Blind, M. (2005). Aptamers as tools for target validation. Current Opinion in Chemical Biology, 9, 336–342. doi: 10.1016/j.cbpa.2005.06.011.
  Bouchard, P. R., Hutabarat, R. M., & Thompson, K. M. (2010). Discovery and development of therapeutic aptamers. Annual Review of Pharmacology and Toxicology, 50, 237–257. doi: 10.1146/annurev.pharmtox.010909.105547.
  Bramsen, J. B., Laursen, M. B., Nielsen, A. F., Hansen, T. B., Bus, C., Langkjaer, N., … Kjems, J. (2009). A large‐scale chemical modification screen identifies design rules to generate siRNAs with high activity, high stability and low toxicity. Nucleic Acids Research, 37, 2867–2881. doi: 10.1093/nar/gkp106.
  Deleavey, G. F., & Damha, M. J. (2012). Designing chemically modified oligonucleotides for targeted gene silencing. Chemistry & Biology, 19, 937–954. doi: 10.1016/j.chembiol.2012.07.011.
  Derrick, W. B., Greef, C. H., Caruthers, M. H., & Uhlenbeck, O. C. (2000). Hammerhead cleavage of the phosphorodithioate linkage. Biochemistry, 39, 4947–4954. doi: 10.1021/bi000146a.
  Elbashir, S. M., 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.
  Fennewald, S. M., Scott, E. P., Zhang, L., Yang, X., Aronson, J. F., Gorenstein, D. G., … Herzog, N. K. (2007). Thioaptamer decoy targeting of AP‐1 proteins influences cytokine expression and the outcome of arenavirus infections. Journal of General Virology, 88, 981–990. doi: 10.1099/vir.0.82499‐0.
  Ferguson, M. R., Rojo, D. R., Somasunderam, A., Thiviyanathan, V., Ridley, B. D., Yang, X., & Gorenstein, D. G. (2006). Delivery of double‐stranded DNA thioaptamers into HIV‐1 infected cells for antiviral activity. Biochemical and Biophysical Research Communications, 344, 792–797. doi: 10.1016/j.bbrc.2006.03.201.
  Gallagher, S. R. (2012). One‐dimensional SDS gel electrophoresis of proteins. Current Protocols in Molecular Biology, 97, 10.2A.1‐10.2A.44. doi: 10.1002/0471142727.mb1002as97.
  Greef, C. H., Seeberger, P. H., & Caruthers, M. H. (1996). Synthesis of phosphorodithioate RNA by the H‐phosphonothioate method. Tetrahedron Letters, 37, 4451–4454. doi: 10.1016/0040‐4039(96)00882‐9.
  Hecht, A. H., Sommer, G. J., Durland, R. H., Yang, X., Singh, A. K., & Hatch, A. V. (2010). Aptamers as affinity reagents in an integrated electrophoretic lab‐on‐a‐chip platform. Analytical Chemistry, 82, 88–13–8820. doi: 10.1021/ac101106m.
  Jain, K. K. (2004). RNAi and siRNA in target validation. Drug Discovery Today, 9, 307–309. doi: 10.1016/S1359‐6446(04)03050‐8.
  Keefe, A. D., Pai, S., & Ellington, A. (2010). Aptamers as therapeutics. Nature Reviews, 9, 537–550. doi: 10.1038/nrd3141.
  Lebedev, A. V., & Wickstrom, E. (1996). The chirality problem in P‐substituted oligonucleotides. Perspectives in Drug Discovery, 4, 17–40. doi: 10.1007/BF02172106.
  Li, N. S., Frederiksen, J. K., & Piccirilli, J. A. (2012). Automated solid‐phase synthesis of RNA oligonucleotides containing a nonbridging phosphorodithioate linkage via phosphorothioamidites. Journal of Organic Chemistry, 77, 9889–9892. doi: 10.1021/jo301834p.
  Marshall, W. S., & Caruthers, M. H. (1993). Phosphorodithioate DNA as a potential therapeutic drug. Science, 259, 1564–1570. doi: 10.1126/science.7681216.
  Melo, S. A., & Esteller, M. (2011). A precursor microRNA in a cancer cell nucleus: Get me out of here! Cell Cycle, 10, 922–925. doi: 10.4161/cc.10.6.15119.
  Osborne, S. E., & Ellington, A. D. (1997). Nucleic acid selection and the challenge of combinatorial chemistry. Chemical Reviews, 97, 349–370. doi: 10.1021/cr960009c.
  Pallan, P., Yang, X., Sierant, M., Abeydeera, N., Hassell, T., Martinez, C., … Egli, M. (2014). Crystal structure, stability and Ago2 affinity of phosphorodithioate‐modified RNAs. RSC Advances, 4, 64901–64904. doi: 10.1039/C4RA10986D.
  Pendergrast, P. S., Marsh, H. N., Grate, D., Healy, J. M., & Stanton, M. (2005). Nucleic acid aptamers for target validation and therapeutic applications. Journal of Biomolecular Techniques, 16, 224–234.
  Petersen, K. H., & Nielsen, J. (1990). Chemical synthesis of dimer ribonucleotides containing internucleotidic phosphorodithioate linkages. Tetrahedron Letters, 31, 911–914. doi: 10.1016/S0040‐4039(00)94661‐6.
  Pushparaj, P. N., Aarthi, J. J., Manikandan, J., & Kumar, S. D. (2008). siRNA, miRNA, and shRNA: In vivo applications. Journal of Dental Research, 87, 992–1003. doi: 10.1177/154405910808701109.
  Sierant, M., Yang, X., Janicka, M., Li, N., Martinez, C., Hassell, T., & Nawrot, B. (2011). SiRNA with phosphorodithioate modification. Collection Symposium Series, XVth Symposium on Chemistry of Nucleic Acid Compounds, 12, 135–139.
  Sierzchala, A., Okruszek, A., & Stec, W. J. (1996). Oxathiaphospholane method of stereocontrolled synthesis of diribonucleoside 3′,5′‐phosphorothioates. Journal of Organic Chemistry, 61, 6713–6716. doi: 10.1021/jo960811e.
  Small, E. M., & Olson, E. N. (2011). Pervasive roles of microRNAs in cardiovascular biology. Nature, 469, 336–342. doi: 10.1038/nature09783.
  Soutschek, J., Akinc, A., Bramlage, B., Charisse, K., Constien, R., Donoghue, M., … Vornlocher, H. P. (2004). Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs. Nature, 432, 173–178. doi: 10.1038/nature03121.
  Stec, W. J., & Wilk, A. (1994). Stereocontrolled synthesis of oligonucleoside phosphorothioates. Angewandte Chemie (International Edition), 33, 709–722. doi: 10.1002/anie.199407091
  Tuerk, C., & 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.
  Verma, S., & Eckstein, F. (1998). Modified oligonucleotides: Synthesis and strategy for users. Annual Review of Biochemistry, 67, 99–134. doi: 10.1146/annurev.biochem.67.1.99.
  Vortler, L. C., & Eckstein, F. (2000). Phosphorothioate modification of RNA for stereochemical and interference analyses. Methods in Enzymology, 317, 74–91. doi: 10.1016/S0076‐6879(00)17007‐7.
  Wada, T., Fujiwara, S., Sato, T., Oka, N., & Saigo, K. (2004). Stereocontrolled synthesis of phosphorothioate RNA by the oxazaphospholidine approach. Nucleic Acids Symposium Series, 48, 57–58. doi: 10.1093/nass/48.1.57.
  Wang, H., Yang, X., Bowick, G. C., Herzog, N. K., Luxon, B. A., Lomas, L. O., & Gorenstein, D. G. (2006). Identification of proteins bound to a thioaptamer probe on a proteomics array. Biochemical and Biophysical Research Communications, 347, 586–593. doi: 10.1016/j.bbrc.2006.06.132.
  Wiesler, W. T., & Caruthers, M. H. (1996). Synthesis of phosphorodithioate DNA via sulfur‐linked, base‐labile protecting groups. Journal of Organic Chemistry, 61, 4272–4281. doi: 10.1021/jo960274y.
  Wu, S. Y., Yang, X., Gharpure, K. M., Hatakeyama, H., Egli, M., McGuire, M. H., … Sood, A. K. (2014). 2′‐OMe‐phosphorodithioate‐modified siRNAs show increased loading into the RISC complex and enhanced anti‐tumour activity. Nature Communications, 5, 3459. doi: 10.1038/ncomms4459.
  Yang, X. (2016). Solid‐phase synthesis of oligodeoxynucleotide analogs containing phosphorodithioate linkages. Current Protocols in Nucleic Acid Chemistry, 66, 4.71.1–4.71.14. doi: 10.1002/cpnc.13.
  Yang, X., Fennewald, S., Luxon, B. A., Aronson, J., Herzog, N. K., & Gorenstein, D. G. (1999). Aptamers containing thymidine 3′‐O‐phosphorodithioates: Synthesis and binding to nuclear factor‐ κB. Bioorganic & Medicinal Chemistry Letters, 9, 3357–3362. doi: 10.1016/S0960‐894X(99)00600‐9.
  Yang, X., & Gorenstein, D. G. (2004). Progress in thioaptamer development. Current Drug Targets, 5, 705–715. doi: 10.2174/1389450043345074.
  Yang, X., Hodge, R. P., Luxon, B. A., Shope, R., & Gorenstein, D. G. (2002). Separation of synthetic oligonucleotide dithioates from monothiophosphate impurities by anion‐exchange chromatography on a mono‐q column. Analytical Biochemistry, 306, 92–99. doi: 10.1006/abio.2001.5694.
  Yang, X., Li, N., & Gorenstein, D. G. (2011). Strategies for the discovery of therapeutic Aptamers. Expert Opinion on Drug Discovery, 6, 75–87. doi: 10.1517/17460441.2011.537321.
  Yang, X., & Mierzejewski, E. (2010). Synthesis of nucleoside and oligonucleoside dithiophosphates. New Journal of Chemistry, 34, 805–819. doi: 10.1039/b9nj00618d.
  Yang, X., Sierant, M., Janicka, M., Peczek, L., Martinez, C., Hassell, T., … Nawrot, B. (2012). Gene silencing activity of siRNA molecules containing phosphorodithioate substitutions. ACS Chemical Biology, 7, 1214–1220. doi: 10.1021/cb300078e.
  Yang, X., Wang, H., Beasley, D., Volk, D., Zhao, X., Luxon, B., … Gorenstein, D. (2006). Selection of thioaptamers for diagnostics and therapeutics. Annals of the New York Academy of Sciences, 1082, 116–119. doi: 10.1196/annals.1348.065.
  Yang, X. B., Sierzchala, A., Misiura, K., Niewiarowski, W., Sochacki, M., Stec, W. J., & Wieczorek, M. W. (1998). The first stereocontrolled solid‐phase synthesis of di‐, tri‐, and tetra[adenosine (2′,5′) phosphorothioate]s. Journal of Organic Chemistry, 63, 7097–7100. doi: 10.1021/jo980522l.
  Zhou, W., Huang, P. J., Ding, J., & Liu, J. (2014). Aptamer‐based biosensors for biomedical diagnostics. Analyst, 139, 2627–2640. doi: 10.1039/c4an00132j.
Key References
  Yang, X., Sierant, M., Janicka, M., Peczek, L., Martinez, C., Hassell, T., Li, N., Li, X., Wang, T., & Nawrot, B. (2012). See above.
  This reference summarizes the methods for synthesis of PS2‐RNAs, as well as the synthesis of PS2‐RNA building blocks, the RNA‐thiophosphoramidites.
  Wiesler, W.T., & Caruthers, M. H. (1996). See above.
  This reference describes the detailed procedure for synthesis of DNA‐thiophosphoramidites. The procedure can be referenced for synthesis of RNA‐thiophosphoramidites.
  Yang, X., et al., (2002). See above.
  This reference describes the detailed method to purify the PS2‐DNAs. The method can be used for isolating PS2‐RNA in high purity.
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