Oligodeoxynucleotide Containing S‐Functionalized 2′‐Deoxy‐6‐Thioguanosine: Facile Tools for Base‐Selective and Site‐Specific Internal Modification of RNA

Shigeki Sasaki1, Kazumitsu Onizuka1, Yosuke Taniguchi1

1 Kyushu University, Maidashi, Higashi‐ku, Fukuoka, Japan
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.49
DOI:  10.1002/0471142700.nc0449s48
Online Posting Date:  March, 2012
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Abstract

Chemically modified oligonucleotides play a significant role for genomic research. Modified nucleosides, such as with a fluorescent dye, can be obtained by chemical synthesis. Site‐specifically modified long nucleic acids are obtained by ligation of chemically modified short oligonucleotides with enzyme, photochemistry, or catalytic DNA. The functionality‐transfer ODN (FT‐ODN), which contains 2′‐deoxy‐6‐thioguanosine (6‐thio‐dG) functionalized with the 2‐methyliden‐1,3‐diketone group, is hybridized with the target RNA to trigger the selective functionalization of the 4‐amino group of the cytosine base at pH 7 or the 2‐amino group of the guanine base at pH 9.4 or at pH 7.4 in the presence of NiCl2. In particular, the functionality‐transfer reaction (FTR) under the alkaline conditions or neutral conditions in the presence of NiCl2 proceeds rapidly and selectively to lead to the modification of the target guanine. The transfer reaction of the acetylene‐containing diketone group produces the acetylene‐modified RNA, which can be subjected to the Cu(I)‐catalyzed “click chemistry” with a variety of azide compounds for highly specific, internal modification of RNA. Curr. Protoc. Nucleic Acid Chem. 48:4.49.1‐4.49.16. © 2012 by John Wiley & Sons, Inc.

Keywords: 2′‐deoxy‐6‐thioguanosine; RNA modification; functionality transfer; click chemistry

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Dimethoxytrityl‐Protected Phophoramidite of S‐Protected 2′‐Deoxy‐6‐Thioguanosine from 2′‐Deoxyguanosine
  • Basic Protocol 2: Preparation of 3‐Chloromethylene‐4‐Phenylbutane‐2,4‐Dione
  • Basic Protocol 3: Preparation of 2′‐Deoxy‐6‐Thioguanosine Containing Oligodeoxynucleotides and Its Functionalization with 3‐Chloromethylene‐4‐Phenylbutane‐2,4‐Dione
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of Dimethoxytrityl‐Protected Phophoramidite of S‐Protected 2′‐Deoxy‐6‐Thioguanosine from 2′‐Deoxyguanosine

  Materials
  • tert‐butyldimethylsilyl chloride (Sigma)
  • 2′‐deoxyguanosine
  • Imidazole
  • N,N‐Dimethylformamide (DMF), anhydrous
  • Ethyl acetate (AcOEt)
  • Saturated NaCl (brine)
  • Dry acetonitrile (CH 3CN)
  • Dry dichloromethane (CH 2Cl 2)
  • Dry triethylamine (Et 3N)
  • 2‐Mesitylenesulfonyl chloride
  • 4‐Dimethylaminopyridine (DMAP)
  • Argon
  • N‐Methylpyrrolidine
  • 2‐Ethylhexyl 3‐mercaptopropionate
  • KH 2PO 4
  • Pyridine
  • Molecular sieves 4Å
  • 1‐Hydroxybenzotriazole
  • Phenoxyacetyl chloride
  • Tetrabutylammonium fluoride (TBAF)
  • Dry tetrahydrofurane (THF)
  • Dimethoxytrityl chloride (DMTrCl)
  • Methanol (MeOH)
  • Diisopropylethylamine
  • N,N‐diisopropylchlorophosphoramidite
  • Hexane
  • 200‐ and 300‐mL round‐bottom flasks
  • Silica gel 60N (spherical, neutral, 63 to 210 µm, Kanto Chemical)
  • Silica gel TLC plate Kiselgel 60F 254 (0.2 mm, Merck)
  • Rotary evaporator
  • Celite pad
  • Vacuum
  • Additional reagents and equipment for TLC ( appendix 3D) and column chromatography ( appendix 3E)

Basic Protocol 2: Preparation of 3‐Chloromethylene‐4‐Phenylbutane‐2,4‐Dione

  Materials
  • Benzoylacetone
  • Ethyl orthoformate
  • Acetic anhydride
  • Argon
  • 10% aq. HCl
  • Tetrahydrofuran (THF)
  • Ethyl acetate (AcOEt)
  • Brine (saturated NaCl)
  • Sodium sulfate (Na 2SO 4)
  • Thionyl chloride (SOCl 2)
  • Toluene
  • 3‐Butyn‐2‐one (Sigma‐Aldrich)
  • Dimethyl sulfide
  • S.18 (Austin et al., )
  • Dichloromethane (CH 2Cl 2)
  • TiCl 4 in dry CH 2Cl 2 solution (Sigma‐Aldrich)
  • Saturated aqueous NaHCO 3 solution
  • Dess‐Martin periodinane
  • Sodium thiosulfate (Na 2S 2O 3)
  • 5‐, 10‐, and 50‐mL round‐bottom flasks
  • Rotary evaporator
  • Celite pad
  • Additional reagents and equipment for TLC ( appendix 3D) and column chromatography ( appendix 3E)

Basic Protocol 3: Preparation of 2′‐Deoxy‐6‐Thioguanosine Containing Oligodeoxynucleotides and Its Functionalization with 3‐Chloromethylene‐4‐Phenylbutane‐2,4‐Dione

  Materials
  • S.15 (see protocol 1)
  • 1 µmol CPG column (Glen Research)
  • 1,8‐Diazabicyclo[5.4.0]undec‐7‐ene (DBU)
  • Acetonitrile (CH 3CN)
  • Dichloromethane (CH 2Cl 2)
  • NaOH
  • NaSH (Wako)
  • HPLC solvent, triethylammonium acetate buffer (TEAA; 0.1 M, pH 7.0)
  • Acetic acid (AcOH)
  • Carbonate buffer at pH 10.0: prepared with 27.5 mL 0.1 M Na 2CO 3 and 22.5 mL NaCl of 0.1 M NaHCO 3
  • S.17or S.19 (see protocol 2)
  • RNA3(rC) (Gene Design)
  • RNaseOUT (recombinant RNase inhibitor; Invitrogen)
  • MES buffer at pH 7.0: 0.5 M sodium morpholinoethanesuflonate (MES) is adjusted to pH 7.0 with 0.1 M NaOH
  • RNA5(rG) (Gene Design)
  • RNA3(rG) (Gene Design)
  • Nickel chloride (NiCl 2)
  • Copper sulfate (CuSO 4)
  • Sodium ascorbate
  • Tris[(1‐benzyl‐1H‐1,2,3‐triazol‐4‐yl)methyl]amine (TBTA; Sigma‐Aldrich)
  • Methoxypolyethylene glycol azide 5000 (PEG5000‐N 3; Sigma‐Aldrich)
  • Dimethyl sulfoxide (DMSO)
  • Automated synthesizer
  • HPLC column, Nacalai Tesque: COSMOSIL 5C18‐AR‐II, 10 × 250 mm
  • HPLC column, SHISEIDO C18, 4.6 × 250 mm
  • 600‐µL microtubes
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Figures

Videos

Literature Cited

Literature Cited
   Ali, M.M., Alam, M.R., Kawasaki, T., Nakayama, S., Nagatsugi, F., and Sasaki, S. 2004. Sequence‐ and base‐specific delivery of nitric oxide to cytidine and 5‐methylcytidine leading to efficient deamination. J. Am. Chem. Soc. 126:8864‐8865.
   Austin, W.B., Bilow, N., Kelleghan, W.J., and Lau, K.S. 1981. Facile synthesis of ethynylated benzoic acid derivatives and aromatic compounds via ethynyltrimethylsilanet. J. Org. Chem. 46:2280‐2286.
   Baum, D.A. and Silverman, S.K. 2007. Deoxyribozyme‐catalyzed labeling of RNA. Angew. Chem. Int. Ed. 46:3502‐3504.
   Chow, C.S., Mahto, S.K., and Lamichhane, T.N. 2008. Combined approaches to site‐specific modification of RNA. ACS Chem. Biol. 3:30‐37.
   Herdewijn, P. (ed.) 2008. Modified Nucleosides in Biochemistry, Biotechnology and Medicine. WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim.
   Jao, C.Y. and Salic, A. 2008. Exploring RNA transcription and turnover in vivo by using click chemistry. Proc. Natl. Acad. Sci. U.S.A. 105:15779‐15784.
   Liu, J. and Taylor, J.S. 1998. Template‐directed photoligation of oligodeoxyribonucleotides via 4‐thiothymidine. Nucleic Acids Res. 26:3300‐3304.
   Ogino, M., Taya, Y., and Fujimoto, K. 2008. Highly selective detection of 5‐methylcytosine using photochemical ligation. Chem. Commun. 45:5996‐5998.
   Onizuka, K., Taniguchi, Y., and Sasaki, S. 2007. Development of novel thioguanosine analogs with the ability of specific modification of cytidine. Nucleic Acids Symp. Series 51:5‐6.
   Onizuka, K, Taniguchi, Y., and Sasaki, S. 2009a. A New odorless procedure for the synthesis of 2′deoxy‐6‐thioguanosine and its incorporation into oligonucleotides. Nucleosides Nucleotides Nucleic Acids 28:752‐760.
   Onizuka, K., Taniguchi, Y., and Sasaki, S. 2009b. Site‐specific covalent modification of nucleic acids guided by functionality‐transfer oligodeoxynucleotides. Bioconjug. Chem. 20:799‐803.
   Onizuka, K., Taniguchi, Y., and Sasaki, S. 2010a. A new usage of functionalized oligodeoxynucleotide probe for site‐specific modification of a guanine base within RNA. Nucleic Acids Res. 38:1760‐1766.
   Onizuka, K., Taniguchi, Y., and Sasaki, S. 2010b. Activation and alteration of base selectivity by metal cations in the functionality‐transfer reaction for RNA modification. Bioconjug. Chem. 21:1508‐1512.
   Onizuka, K., Shibata, A., Taniguchi, Y., and Sasaki, S. 2011. Pin‐point chemical modification of RNA with diverse molecules through the functionality transfer reaction and copper‐catalyzed azide‐alkyne cycloaddition reaction. Chem. Comm. 47:5004‐5006.
   Pasternak, A. and Wengel, J. 2011. Unlocked nucleic acid‐an RNA modification with broad potential. Org. Biomol. Chem. 9:3591‐3597.
   Sasaki, S., Onizuka, K., and Taniguchi, Y. 2011. The oligodeoxynucleotide probes for the site‐specific modification of RNA. Chem. Soc. Rev. DOI: 10.1039/c1cs15066a.
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