Convenient and Efficient Approach to the Permanent or Reversible Conjugation of RNA and DNA Sequences with Functional Groups

Jacek Cieślak1, Cristina Ausín1, Andrzej Grajkowski1, Serge L. Beaucage1

1 Food and Drug Administration, Bethesda, Maryland
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.52
DOI:  10.1002/0471142700.nc0452s50
Online Posting Date:  September, 2012
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Abstract

The conversion of 3′,5′‐disilylated 2′‐O‐(methylthiomethyl)ribonucleosides to 2′‐O‐(phthalimidooxymethyl)ribonucleosides is achieved in yields of 66% to 94%. Desilylation and dephtalimidation of these ribonucleosides by treatment with NH4F in MeOH produce 2′‐O‐aminooxymethylated ribonucleosides, which are efficient in producing stable and yet reversible 2′‐conjugates upon reaction with 1‐pyrenecarboxaldehyde. Exposure of 2′‐pyrenylated ribonucleosides to 0.5 M tetra‐n‐butylammonium fluoride (TBAF) in THF or DMSO results in the cleavage of their iminoether functions to give the native ribonucleosides along with an innocuous nitrile side product. Conversely, the reaction of 2′‐O‐(aminooxymethyl)uridine with 5‐cholesten‐3‐one leads to a permanent uridine 2′‐conjugate, which is left unreacted when treated with TBAF. The versatility and uniqueness of 2′‐O‐(aminooxymethyl)ribonucleosides is demonstrated by the single or double incorporation of a reversible pyrenylated uridine 2′‐conjugate into an RNA sequence. Furthermore, the conjugation of 2′‐O‐(aminooxymethyl)ribonucleosides with various aldehydes, including those generated from their acetals, is also presented. The preparation of 5′‐O‐(aminooxymethyl)thymidine is also achieved, albeit in modest yields, from the conversion of 5′‐O‐methylthiomethyl‐3′‐O‐(levulinyl)thymidine to 5′‐O‐phthalimidooxymethyl‐3′‐O‐(levuliny)lthymidine followed by hydrazinolysis of both 5′‐phthalimido and 3′‐levulinyl groups. Pyrenylation of the 5′‐O‐(aminooxymethyl)deoxyribonucleoside also provides a reversible 5′‐conjugate that is sensitive to TBAF, thereby further demonstrating the usefulness of 5′‐O‐(aminooxymethyl)deoxyribonucleosides for permanent or reversible modification of DNA sequences. Curr. Protoc. Nucleic Acid Chem. 50:4.52.1‐4.52.36. © 2012 by John Wiley & Sons, Inc.

Keywords: 2′‐O‐(aminooxymethyl)ribonucleosides; 5′‐O‐(aminooxymethyl)deoxyribonucleosides; permanent or reversible conjugation; modification of DNA or RNA sequences; ribonucleoside 2′‐conjugates; deoxyribonucleoside 5′‐conjugates

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesis, Purification, and Characterization of 2′‐O‐(Aminooxymethyl)Ribonucleosides
  • Alternate Protocol 1: Synthesis, Purification and Characterization of an Exemplary 5′‐O‐(Aminooxymethyl)Deoxyribonucleoside
  • Basic Protocol 2: Synthesis, Purification, and Characterization of Ribonucleoside 2′‐Conjugates and of a Deoxyribonucleoside 5′‐Conjugate
  • Support Protocol 1: Reversibility of Exemplary 2′‐O‐(Aminooxymethyl)Ribonucleoside Conjugates
  • Basic Protocol 3: Synthesis, Purification, Characterization, and Reversibility of 2′‐Pyrenylated Chimeric RNA Sequences
  • Alternate Protocol 2: Synthesis, Purification, Characterization, and Reversibility of a 5′‐Pyrenylated DNA Sequence
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Synthesis, Purification, and Characterization of 2′‐O‐(Aminooxymethyl)Ribonucleosides

  Materials
  • 3′,5′‐O‐(1,1,3,3‐Tetraisopropyldisiloxane‐1,3‐diyl)uridine (Rasayan, http://www.rasayan.us/)
  • Dimethyl sulfoxide (DMSO; Acros)
  • Glacial acetic acid (AcOH; Acros)
  • Acetic anhydride (Ac 2O; Acros)
  • Drierite (20‐40 mesh; Aldrich)
  • Potassium carbonate, anhydrous (K 2CO 3,Fisher)
  • Tetrahydrofuran (THF; Acros)
  • Pyridine, anhydrous (Acros)
  • Toluene (Acros)
  • Methylene chloride, anhydrous (CH 2CH 2, Fisher)
  • Silica gel (60 Å, 230‐400 mesh; Merck)
  • Methanol (Fisher)
  • Chloroform (CH 3Cl; Fisher)
  • Benzene (Aldrich), dry
  • Dry ice
  • Acetone
  • Sulfuryl chloride (Aldrich)
  • N‐Hydroxyphthalimide (Acros), vacuum dried
  • 1,8‐Diazabicyclo[5.4.0]undec‐7‐ene (DBU; Acros), dry
  • Sodium hydrogen carbonate (NaHCO 3; Fisher)
  • Anhydrous sodium sulfate (Fisher)
  • Ammonium fluoride (Aldrich)
  • N4‐Acetyl‐3′,5′‐O‐(1,1,3,3‐tetraisopropyldisiloxane‐1,3‐diyl)cytidine (Rasayan, http://www.rasayan.us/)
  • Ammonium hydroxide (Aldrich)
  • N6‐Isobutyryl‐3′,5′‐O‐(1,1,3,3‐tetraisopropyldisiloxane‐1,3‐diyl)adenosine (Rasayan, http://www.rasayan.us/)
  • N2‐Phenoxyacetyl‐3′,5′‐O‐(1,1,3,3‐tetraisopropyldisiloxane‐1,3‐diyl)guanosine (Rasayan, http://www.rasayan.us/)
  • Magnetic stirrer and stir bars (VWR)
  • 10‐, 25‐, 50‐, 100‐, 250‐mL round‐bottom flasks (Kontes)
  • 4‐mL screw‐cap glass vials
  • Rubber septa for 14/20‐ and 24/40‐glass joints (Aldrich)
  • 1‐, 3‐, 10‐mL plastic syringes (Becton Dickinson)
  • 21‐G stainless steel syringe needles (Fisher)
  • Heating mantles (VWR)
  • 100‐, 250‐, and 2000‐mL Erlenmeyer flasks (Kimax)
  • 5 × 20–cm glass chromatography columns (Labglass)
  • 2.5 × 7.5–cm TLC plates precoated with a 250‐µm layer of silica gel 60 F 254 (EMD)
  • Chromaflex TLC developing jars (Kontes)
  • Lyophilizer
  • Rotary evaporator (Büchi, http://www.buchi.com/) connected to a vacuum pump (KNF, http://www.knf.com/)
  • High‐vacuum oil pump (Savant)
  • 100‐ and 250‐mL separatory funnels (Kontes)
  • 30‐mL sintered‐glass funnel (coarse porosity, Kontes)
  • 2.5 × 20–cm disposable Flex chromatography columns (Kontes)
  • 50‐mL beaker (optional; Kimax)
  • Heat block (VWR)
  • Additional reagents and equipment for thin‐layer chromatography (TLC; appendix 3D)

Alternate Protocol 1: Synthesis, Purification and Characterization of an Exemplary 5′‐O‐(Aminooxymethyl)Deoxyribonucleoside

  • 3′‐O‐Levulinyl‐2′‐deoxythymidine (Rasayan, http://www.rasayan.us/)
  • Dry argon gas cylinder (Matheson)
  • Hydrazine hydrate (Aldrich)

Basic Protocol 2: Synthesis, Purification, and Characterization of Ribonucleoside 2′‐Conjugates and of a Deoxyribonucleoside 5′‐Conjugate

  Materials
  • Silica gel–purified S.4a ( protocol 1, step 12)
  • Methanol (MeOH; Fisher)
  • Ammonium fluoride (Aldrich)
  • 1‐Pyrenecarboxaldehyde (Aldrich)
  • Methylene chloride (CH 2CH 2; Fisher)
  • Sodium hydrogen carbonate (NaHCO 3, Fisher)
  • Silica gel (60 Å, 230 to 400 mesh; Merck)
  • Chloroform (CHCl 3; Fisher)
  • Ammonium hydroxide (Fisher)
  • Silica gel–purified S.4b (see protocol 1)
  • Silica gel–purified S.4c (see protocol 1)
  • Silica gel–purified S.4d (see protocol 1)
  • Silica gel–purified S.10 (see protocol 2)
  • 5‐Cholesten‐3‐one (Aldrich)
  • D‐(+)Biotin 2‐nitrophenyl ester (Berry & Associates, http://www.berryassoc.com/)
  • Acetonitrile (MeCN; Acros)
  • Aminoacetaldehyde dimethyl acetal (Aldrich)
  • Triethylamine (Et 3N; Aldrich)
  • Hydrochloric acid, concentrated (Fisher)
  • Dansyl chloride (Aldrich)
  • 4‐(Dimethylamino)azobenzene‐4′‐sulfonyl chloride (Aldrich)
  • Iodine (I 2; Aldrich)
  • Acetone (Fisher)
  • Sodium bisulfite (Aldrich)
  • 10‐, 25‐, 50‐, 100‐, 250‐mL round‐bottom flasks (Kontes)
  • Rubber septa for 14/20‐ and 24/40‐glass joints (Aldrich)
  • Magnetic stirrer and stir bars (VWR)
  • 4‐mL screw‐cap glass vials (e.g., Wheaton)
  • Heat block (VWR)
  • 25‐, 100‐, and 250‐mL separatory funnels (Kontes)
  • Rotary evaporator (Büchi, http://www.buchi.com/) connected to a vacuum pump (KNF, http://www.knf.com/)
  • 2.5 × 20–cm disposable Flex chromatography columns (Kontes)
  • 50‐mL beaker (optional)
  • 2.5 × 7.5‐cm TLC plates precoated with a 250‐µm layer of silica gel 60 F 254 (EMD)
  • Chromaflex TLC developing jars (Kontes)
  • Heat gun (VWR)
  • Additional reagents and equipment for thin‐layer chromatography (TLC; appendix 3D)

Support Protocol 1: Reversibility of Exemplary 2′‐O‐(Aminooxymethyl)Ribonucleoside Conjugates

  Materials
  • 2′‐O‐(pyren‐1‐ylmethanimine‐N‐oxymethyl)uridine ( S.11a; see protocol 3, step 5)
  • Tetra‐n‐butylammonium fluoride (TBAF; Aldrich)
  • Tetrahydrofuran (THF; Acros)
  • HPLC buffer A: 0.1 M triethylammonium acetate, pH 7.0 (Applied Biosystems)
  • HPLC buffer B: acetonitrile
  • Biotinylated uridine 2′‐conjugate ( S.16; see protocol 3, step 30)
  • Dansylated uridine 2′‐conjugate ( S.18; see protocol 3, step 39)
  • Dansylated uridine 2′‐conjugate ( S.20; see protocol 3, text between steps 41 and 42)
  • Dabsylated cytidine 2′‐conjugate ( S.22; see protocol 3, step 47)
  • 4‐mL screw‐cap glass vials (Wheaton)
  • Heat block (VWR)
  • 5 µm Supelcosil LC‐18S HPLC column (25 cm × 4.6 mm, Supelco)
  • Additional reagents and equipment for RP‐HPLC (unit 10.5)

Basic Protocol 3: Synthesis, Purification, Characterization, and Reversibility of 2′‐Pyrenylated Chimeric RNA Sequences

  Materials
  • 2′‐O‐(pyren‐1‐ylmethanimine‐N‐oxymethyl)uridine ( S.11a; see protocol 3, step 5)
  • Pyridine (Acros), anhydrous
  • 4,4′‐Dimethoxytrityl chloride (Chem‐Impex International, http://www.chemimpex.com/)
  • Methylene chloride (CH 2CH 2; Fisher)
  • Sodium hydrogen carbonate (NaHCO 3; Aldrich)
  • Anhydrous sodium sulfate (Na 2SO 4;Fisher)
  • Methanol (Fisher)
  • Silica gel (60 Å, 230 to 400 mesh; Merck)
  • Triethylamine (Et 3N; Aldrich)
  • Chloroform (CHCl 3; Fisher)
  • Dry argon gas cylinder (Matheson)
  • 2‐Cyanoethyl N,N‐diisopropylchlorophosphoramidite (Aldrich)
  • Benzene (C 6H 6; Aldrich)
  • Hexane (Fisher)
  • Dry ice
  • Acetone
  • Monomers and reagents for oligonucleotide synthesis:
    • N4‐Acetyl‐5′‐O‐(4,4′‐dimethoxytrityl)‐3′‐O‐[(N,N‐diisopropylamino)(2‐cyanoethyloxy)]phosphinyl‐2′‐O‐(tert‐butyldimethylsilyl)cytidine (ChemGenes)
    • N6‐Isobutyryl‐5′‐O‐(4,4′‐dimethoxytrityl)‐3′‐O‐[(N,N‐diisopropylamino)(2‐cyanoethyloxy)]phosphinyl‐2′‐O‐(tert‐butyldimethylsilyl)adenosine (ChemGenes)
    • 5′‐O‐(4,4′‐Dimethoxytrityl)‐3′‐O‐[(N,N‐diisopropylamino)(2‐cyanoethyloxy)] phosphinyl‐2′‐O‐(tert‐butyldimethylsilyl)uridine (ChemGenes)
    • N2‐Phenoxyacetyl‐5′‐O‐(4,4′‐dimethoxytrityl)‐3′‐O‐[(N,N‐diisopropylamino)(2‐cyanoethyloxy)]phosphinyl‐2′‐O‐(tert‐butyldimethylsilyl)guanosine (ChemGenes)
    • 5′‐O‐(4,4′‐Dimethoxytrityl)‐3′‐O‐[(N,N‐diisopropylamino) (2‐cyanoethyloxy)] phosphinyl‐2′‐O‐(pyren‐1‐ylmethanimine‐N‐oxymethyl)uridine ( S.24)
    • 0.45 M 1H‐Tetrazole in MeCN (Glen Research)
    • 0.25 M 5‐Benzylthio‐1H‐tetrazole (Glen Research)
    • Cap A solution: acetic anhydride in tetrahydrofuran/pyridine (Glen Research)
    • Cap B solution: 1‐methylimidazole in tetrahydrofuran (Glen Research)
    • Deblocking solution: 3% trichloroacetic acid in CH 2Cl 2 (Glen Research)
    • Oxidation solution: 0.02 M iodine in THF/pyridine/water (Glen Research)
  • Ammonium hydroxide (Aldrich)
  • Dimethyl sulfoxide (DMSO; Acros)
  • Triethylamine trihydrofluoride (Et 3N3HF) (Aldrich)
  • 2 M triethylammonium acetate (TEAA) buffer pH 7.0 (Applied Biosystems)
  • Acetonitrile (MeCN; Acros)
  • Diethylpyrocarbonate (DEPC)‐treated water (Research Genetics)
  • Tetra‐n‐butylammonium fluoride (TBAF; Aldrich)
  • 25‐, 100‐, 250‐mL round‐bottom flasks (Kontes)
  • Rubber septa for 14/20‐ and 24/40‐glass joints (Aldrich)
  • Rotary evaporator (Büchi, http://www.buchi.com/) connected to a vacuum pump (KNF, http://www.knf.com/)
  • 1‐, 3‐, and 10‐mL plastic syringes (B‐D)
  • 21‐G stainless steel syringe needles (Fisher)
  • 25‐mL separatory funnels (Kontes)
  • 2.5 × 20–cm disposable Flex chromatography columns (Kontes)
  • Magnetic stirrer/hot plate and stir bars (VWR)
  • 2.5 × 7.5‐cm TLC plates precoated with a 250‐µm layer of silica gel 60 F 254 (EMD)
  • Chromaflex TLC developing jars (Kontes)
  • Filter paper, no. 1 (Whatman)
  • Automated DNA/RNA synthesizer (Applied Biosystems Model 392)
  • Synthesis columns with long‐chain alkylamine controlled‐pore glass (LCAA‐CPG, 500 Å) support loaded with 5′‐O‐(4,4′‐dimethoxytrityl)‐2′‐deoxythymidine covalently bound through a 3′‐O‐hemisuccinate linker (Glen Research)
  • 4‐mL screw‐cap glass vials (Fisher)
  • 5 µm Supelcosil LC‐18S HPLC column (25 cm × 4.6 mm, Supelco)
  • Speedvac evaporator connected to a vacuum pump
  • PD‐10 column (Sephadex G‐25M, GE Healthcare)
  • UV spectrophotometer
  • Lyophilizer (optional)
  • Additional reagents and equipment for thin‐layer chromatography (TLC; appendix 3D), oligonucleotide synthesis ( appendix 3C), and RP‐HPLC analyses (unit 10.5)

Alternate Protocol 2: Synthesis, Purification, Characterization, and Reversibility of a 5′‐Pyrenylated DNA Sequence

  • 5′‐O‐(pyren‐1‐ylmethanimine‐N‐oxymethyl)‐2′‐deoxythymidine ( S.12; see protocol 3, step 16)
  • 50‐mL separatory funnels (Kontes)
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Figures

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Literature Cited

Literature Cited
   Beaucage, S.L. 1999. Attachment of reporter and conjugate groups to DNA. In Comprehensive Natural Products Chemistry: DNA and Aspect of Molecular Biology, Vol. 7 (D. Barton, H. Nakanishi, O. Meth‐Cohn, E.T. Kool, eds.) pp. 153‐249. Elsevier, Oxford, United Kingdom.
   Beaucage, S.L. 2008. Solid‐phase synthesis of siRNA oligonucleotides. Curr. Opin. Drug Disc. Devel. 11:203‐216.
   Cieślak, J., Grajkowski, A., Ausín, C., Gapeev, A., and Beaucage, S.L. 2012. Permanent or reversible conjugation of 2′‐O‐ or 5′‐O‐aminooxymethylated nucleosides with functional groups as a convenient and efficient approach to the modification of RNA and DNA sequences. Nucleic Acids Res. 40:2312‐2329.
   Clark, J.H. 1980. Fluoride ion as a base in organic synthesis. Chem. Rev. 80:429‐452.
   Cook, P.D. 1998. Second generation antisense oligonucleotides: 2′‐Modifications. In Annual Report in Medicinal Chemistry, Vol. 33 (J.A. Bristol, ed.) pp. 313‐325. Academic Press, San Diego, California.
   Defrancq, E. and Lhomme, J. 2001. Use of an aminooxy linker for the functionalization of oligodeoxyribonucleotides. Bioorg. Med. Chem. Lett. 11:931‐933.
   Fang, S. and Bergstrom, D.E. 2003a. Reversible biotinylation phosphoramidite for 5′‐end‐labeling, phosphorylation, and affinity purification of synthetic oligonucleotides. Bioconjug. Chem. 14:80‐85.
   Fang, S. and Bergstrom, D.E. 2003b. Fluoride‐cleavable biotinylation phosphoramidite for 5′‐end‐labeling and affinity purification of synthetic oligonucleotides. Nucleic Acids Res. 31:708‐715.
   Fang, S. and Bergstrom, D.E. 2004. Reversible 5′‐end biotinylation and affinity purification of synthetic RNA. Tetrahedron Lett. 45:7987‐7990.
   Fang, S. and Fueangfung, S. 2010. Scalable synthetic oligodeoxynucleotide purification with use of a catching by polymerization, washing, and releasing approach. Org. Lett. 12:3720‐3723.
   Forget, D., Boturyn, D., Defrancq, E., Lhomme, J., and Dumy, P. 2001a. Highly efficient synthesis of peptide—oligonucleotide conjugates: Chemoselective oxime and thiazolidine formation. Chem. Eur. J. 7:3976‐3984.
   Forget, D., Renaudet, O., Defrancq, E., and Dumy, P. 2001b. Efficient preparation of carbohydrate–oligonucleotide conjugates (COCs) using oxime bond formation. Tetrahedron Lett. 42:7829‐7832.
   Forget, D., Renaudet, O., Boturyn, D., Defrancq, E., and Dumy, P. 2001c. 3′‐Oligonucleotides conjugation via chemoselective oxime bond formation. Tetrahedron Lett. 42:9171‐9174.
   Griffey, R., Lesnik, E., Freier, S., Sanghvi, Y.S., Teng, K., Kawasaki, A., Guinosso, C., Wheeler, P., Mohan, V., and Cook, P.D. 1994. New twists on nucleic acids structural properties of modified nucleosides incorporated into oligonucleotides. In Carbohydrate Modifications in Antisense Research (Y.S. Sanghvi and P.D. Cook, eds.) pp. 212‐224. American Chemical Society, Washington, D.C.
   Hilvert, D. 1994. Chemical synthesis of proteins. Chem. Biol. 1:201‐203.
   Katajisto, J., Virta, P., and Lönnberg, H. 2004. Solid‐phase synthesis of multiantennary oligonucleotide glycoconjugates utilizing on‐support oximation. Bioconjug. Chem. 15:890‐896.
   Kawasaki, A.M., Casper, M.D., Prakash, T.P., Manalili, S., Sasmor, H., Manoharan, M., and Cook, P.D. 1999. Synthesis, hybridization, and nuclease resistance properties of 2′‐O‐aminooxyethyl (2′‐O‐AOE) modified oligonucleotides. Tetrahedron Lett. 40:661‐664.
   Landini, D., Maia, A., and Rampoldi, A. 1989. Dramatic effect of the specific solvation on the reactivity of quaternary ammonium fluorides and poly(hydrogen fluorides), (HF)n• F− in media of low polarity. J.Org. Chem. 54:328‐332.
   Manoharan, M. 1999. 2′‐Carbohydrate modifications in antisense oligonucleotide therapy: Importance of conformation, configuration and conjugation. Biochim. Biophys. Acta 1489:117‐130.
   Morvan, F., Sanghvi, Y.S., Perbost, M., Vasseur, J.‐J., and Bellon, L. 1996. Oligonucleotide mimics for antisense therapeutics: Solution phase and automated solid‐support synthesis of MMI linked oligomers. J. Am. Chem. Soc. 118:255‐256.
   Parey, N., Baraguey, C., Vasseur, J.‐J., and Debart, F. 2006. First evaluation of acyloxymethyl or acylthiomethyl groups as biolabile 2′‐O‐protection of RNA. Org. Lett. 8:3869‐3872.
   Rastogi, H. and Usher, D.A. 1995. A new 2′‐hydroxyl protecting group for the automated synthesis of oligoribonucleotides. Nucleic Acids Res. 23:4872‐4877.
   Rodriguez, E.C., Winans, K.A., King, D.S. and Bertozzi, C.R. 1997. A strategy for the chemoselective synthesis of O‐linked glycopeptides with native sugar‐peptide linkages. J. Am. Chem. Soc. 119:9905‐9906.
   Rose, K. 1994. Facile synthesis of homogeneous artificial proteins. J. Am. Chem. Soc. 116:30‐33.
   Rozners, E. 2006. Carbohydrate chemistry for RNA interference: Synthesis and properties of RNA analogues modified in sugar‐phosphate backbone. Curr. Org. Chem. 10:675‐692.
   Salo, H., Virta, P., Hakala, H., Prakash, T.P., Kawasaki, A.M., Manoharan, M., and Lönnberg, H. 1999. Aminooxy functionalized oligonucleotides: Preparation, on‐support derivatization, and postsynthetic attachment to polymer support. Bioconjug. Chem. 10:815‐823.
   Semenyuk, A., Földesi, A., Johansson, T., Estmer‐Nilsson, C., Blomgren, P., Brännvall, M., Kirsebom, L.A., and Kwiatkowski, M. 2006. Synthesis of RNA using 2′‐O‐DTM protection. J. Am. Chem. Soc. 128:12356‐12357.
   Silverman, S.K. and Cech, T.R. 1999. RNA tertiary folding monitored by fluorescence of covalently attached pyrene. Biochemistry 38:14224‐14237.
   Sun, J., Dong, Y., Cao, L., Wang, X., Wang, S., and Hu, Y. 2004. Highly efficient chemoselective deprotection of O,O‐acetals and O,O‐ketals catalyzed by molecular iodine in acetone. J. Org. Chem. 69:8932‐8934.
   Trevisiol, E., Renard, A., Defrancq, E., and Lhomme, J. 1997. The oxyamino‐aldehyde coupling reaction: An efficient method for the derivatization of oligonucleotides. Tetrahedron Lett. 38:8687‐8690.
   Ti, G.S, Gaffney, B.L., and Jones, R.A. 1982. Transient protection: Efficient one‐flask syntheses of protected deoxynucleosides. J. Am. Chem. Soc. 104:1316‐1319.
   Tomaya, K., Takahashi, M., Minakawa, N., and Matsuda, A. 2010. Convenient RNA synthesis using a phosphoramidite possessing a biotinylated photocleavable group. Org. Lett. 12:3836‐3839.
   Watts, J.K., Deleavey, G.F., and Damha, M.J. 2008. Chemically modified siRNA: Tools and applications. Drug Disc. Today 13:842‐855.
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